Treatment of multiple sclerosis using placental stem cells

ABSTRACT

The present invention provides methods of immunomodulation using placental stem cells and placental stem cell populations. The invention also provides methods of producing and selecting placental cells and cell populations on the basis of immunomodulation, and compositions comprising such cells and cell populations.

This application is a division of U.S. patent application Ser. No.11/580,588, filed Oct. 13, 2006, which claims benefit of U.S.Provisional Application No. 60/727,004, filed Oct. 13, 2005, and60/835,628, filed Aug. 4, 2006, each of which is hereby incorporated byreference in its entirety.

1. FIELD OF THE INVENTION

The present invention provides methods of using placental stem cells tomodulate the immune system, and immune responses to antigens. Theinvention also provides compounds comprising placental stem cells foruse in immunomodulation, and methods of transplanting tissues and organscomprising administration of placental stem cells to prevent or inhibitimmune-mediated rejection.

2. BACKGROUND OF THE INVENTION

Human stem cells are totipotential or pluripotential precursor cellscapable of generating a variety of mature human cell lineages. Evidenceexists that demonstrates that stem cells can be employed to repopulatemany, if not all, tissues and restore physiologic and anatomicfunctionality.

Many different types of mammalian stem cells have been characterized.See, e.g., Caplan et al., U.S. Pat. No. 5,486,359 (human mesenchymalstem cells); Boyse et al., U.S. Pat. No. 5,004,681 (fetal and neonatalhematopoietic stem and progenitor cells); Boyse et al., U.S. Pat. No.5,192,553 (same); Beltrami et al., Cell 114(6):763-766 (2003) (cardiacstem cells); Forbes et al., J. Pathol. 197(4):510-518 (2002) (hepaticstem cells). Umbilical cord blood, and total nucleated cells derivedfrom cord blood, have been used in transplants to restore, partially orfully, hematopoietic function in patients who have undergone ablativetherapy.

The placenta is a particularly attractive source of stem cells. Becausemammalian placentas are plentiful and are normally discarded as medicalwaste, they represent a unique source of medically-useful stem cells.The present invention provides such isolated placental stem cells,populations of the placental stem cells, and methods of using the same.

3. SUMMARY OF THE INVENTION

The present invention provides methods of immunosuppression usingpluralities of placental stem cells or umbilical cord stem cells,populations of placental stem cells or umbilical cord stem cells, andcompositions comprising and/or produced by the stem cells. The presentinvention also provides compositions, including compositions comprisingplacental stem cells or umbilical cord stem cells, havingimmunosuppressive properties. The invention further provides populationsof placental cells or umbilical cord stem cells selected on the basis oftheir ability to modulate an immune response, and compositions havingimmunomodulatory properties.

In one aspect, the invention provides a method of suppressing orreducing an immune response comprising contacting a plurality of immunecells with a plurality of placental stem cells for a time sufficient forsaid placental stem cells to detectably suppress an immune response,wherein said placental stem cells detectably suppress T cellproliferation in a mixed lymphocyte reaction (MLR) assay. In a specificembodiment, said placental stem cells: express CD200 and HLA-G; expressCD73, CD105, and CD200; express CD200 and OCT-4; express CD73, CD105,and HLA-G; express CD73 and CD105 and facilitate the formation of one ormore embryoid-like bodies in a population of placental cells thatcomprises the plurality of placental stem cells when said population iscultured under conditions that allow formation of embryoid-like bodies;and/or express OCT-4 and facilitate the formation of one or moreembryoid-like bodies in a population of placental cells that comprisesthe plurality of placental stem cells, when said population is culturedunder conditions that allow formation of embryoid-like bodies. Inanother specific embodiment, said plurality of immune cells are T cellsor NK (natural killer) cells. In a more specific embodiment, said Tcells are CD4⁺ T cells. In another more specific embodiment, said Tcells are CD8⁺ T cells. In another specific embodiment, said contactingis performed in vitro. In another specific embodiment, said contactingis performed in vivo. In a more specific embodiment, said in vivocontacting is performed in a mammalian subject, e.g., a human subject.In another more specific embodiment, said contacting comprisesadministering said placental cells intravenously, intramuscularly, orinto an organ in said subject (e.g., a pancreas). The method ofsuppressing an immune response, particularly in vivo, can additionallycomprise administering (e.g., to a mammal), e.g., an anti-macrophageinflammatory protein (MIP)-1α or anti-MIP-1β antibody to said subject,wherein said antibody is administered in an amount sufficient to cause adetectable drop in the amount of MIP-1α or anti-MIP-1β, e.g., in bloodfrom said subject.

In a more specific embodiment of the method, said placental stem cellsthat express CD200 and HLA-G also express CD73 and CD105, that is, areCD73⁺ and CD105⁺. In another specific embodiment, said placental cellsare CD34⁻, CD38⁻ or CD45⁻. In a more specific embodiment, said placentalstem cells are CD34⁻, CD38⁻, CD45⁻, CD73⁺ and CD105⁺. In anotherspecific embodiment, said plurality of placental stem cells facilitatesthe development of one or more embryoid-like bodies from a population ofisolated placental cells comprising the placental stem cells when saidpopulation is cultured under conditions that allow formation ofembryoid-like bodies.

In another more specific embodiment of the method, said placental stemcells that express CD73, CD105, and CD200 are also HLA-G⁺. In anotherspecific embodiment, said placental stem cells are CD34⁻, CD38⁻ orCD45⁻. In another specific embodiment, said placental stem cells areCD34⁻, CD38⁻ and CD45⁻. In a more specific embodiment, said placentalstem cells are CD34⁻, CD38⁻, CD45⁻, and HLA-G⁺. In another specificembodiment, said placental stem cells facilitate the development of oneor more embryoid-like bodies from a population of isolated placentalcells comprising the placental stem cells when said population iscultured under conditions that allow formation of embryoid-like bodies.

In another more specific embodiment of the method, said placental stemcells that express CD200 and OCT-4 also express CD73⁺ and CD105⁺. Inanother specific embodiment, said placental stem cells are HLA-G⁺. Inanother specific embodiment, said placental stem cells are CD34⁻, CD38⁻or CD45⁻. In another specific embodiment, said placental stem cells areCD34⁻, CD38⁻ and CD45⁻. In a more specific embodiment, said placentalstem cells are CD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺ and HLA-G⁺. In anotherspecific embodiment, said placental stem cells facilitate the formationof one or more embryoid-like bodies from a population of isolatedplacental cells comprising placental stem cells when said population iscultured under conditions that allow formation of embryoid-like bodies.

In another more specific embodiment, said placental stem cells thatexpress CD73, CD105, and HLA-G are also CD34⁻, CD38⁻ or CD45⁻. Inanother specific embodiment, said placental stem cells are CD34⁻, CD38⁻and CD45⁻. In another specific embodiment, said placental stem cells areOCT-4⁺. In another specific embodiment, said placental stem cells areCD200⁺. In a more specific embodiment, said placental stem cells areCD34⁻, CD38⁻, CD45⁻, OCT-4⁺ and CD200⁺. In another specific embodiment,said stem cells facilitate the formation of one or more embryoid-likebodies from a population of isolated placental cells comprising theplacental stem cells when said population is cultured under conditionsthat allow formation of embryoid-like bodies.

In another more specific embodiment, said placental stem cells thatexpress CD73 and CD105, and facilitate the formation of one or moreembryoid-like bodies in a population of placental cells that comprisethe placental stem cells when said population is cultured underconditions that allow formation of embryoid-like bodies, are also CD34⁻,CD38⁻ or CD45⁻. In another specific embodiment, said placental stemcells are OCT-4⁺. In another specific embodiment, said placental stemcells are CD200⁺. In another specific embodiment, said placental stemcells are OCT-4⁺, CD200⁺, CD34⁻, CD38⁻ and CD45⁻.

In another more specific embodiment, said placental stem cells thatexpress OCT-4, and facilitate the formation of one or more embryoid-likebodies in a population of placental cells that comprise the placentalstem cells when said population is cultured under conditions that allowformation of embryoid-like bodies, are also CD73⁺ and CD105⁺. In anotherspecific embodiment, said placental stem cells are CD34⁻, CD38⁻ andCD45⁻. In another specific embodiment, said placental stem cells areCD200⁺. In another specific embodiment, said placental stem cells areCD73⁺, CD105⁺, CD200⁺, CD34⁻, CD38⁻ and CD45⁻.

In another specific embodiment of the method of reducing or suppressingan immune response, said immune response is graft-versus-host disease.In another specific embodiment, said immune response is an autoimmunedisease, e.g., diabetes, lupus erythematosus, or rheumatoid arthritis.

In another specific embodiment of the method, said plurality of immunecells is also contacted with a plurality of non-placental cells. Suchnon-placental cells can, e.g., comprise CD34⁺ cells. In a more specificembodiment, said CD34⁺ cells are peripheral blood hematopoieticprogenitor cells, cord blood hematopoietic progenitor cells, orplacental blood hematopoietic progenitor cells. In another specificembodiment, said non-placental cells comprise mesenchymal stem cells. Ina more specific embodiment, said mesenchymal stem cells are bonemarrow-derived mesenchymal stem cells. In another specific embodiment,said non-placental cells are contained within an allograft.

The method can employ as many placental stem cells as are required toeffect a detectable suppression of an immune response. For example, theplurality of placental stem cells sued to contact the plurality ofimmune cells can comprise 1×10⁵ placental stem cells, 1×10⁶ placentalstem cells, 1×10⁷ placental stem cells, or 1×10⁸ placental stem cells,or more.

The invention further provides methods of producing cell populationscomprising placental stem cells selected on the basis of their abilityto modulate (e.g., suppress) an immune response. In one embodiment, forexample, the invention provides a method of selecting a placental cellpopulation comprising (a) assaying a plurality of placental cells in amixed lymphocyte reaction (MLR) assay; and (b) selecting said pluralityof placental stem cells if said plurality of placental stem cellsdetectably suppresses CD4⁺ or CD8⁺ T cell proliferation in an MLR (mixedlymphocyte reaction), wherein said placental stem cells: (i) adhere to asubstrate, and (ii) express CD200 and HLA-G, or express CD73, CD105, andCD200, or express CD200 and OCT-4, or express CD73, CD105, and HLA-G, orexpress CD73 and CD105, and facilitate the formation of one or moreembryoid-like bodies in a population of placental cells that comprisethe stem cell, when said population is cultured under conditions thatallow formation of embryoid-like bodies, or express OCT-4, andfacilitate the formation of one or more embryoid-like bodies in apopulation of placental cells that comprise the stem cell, when saidpopulation is cultured under conditions that allow formation ofembryoid-like bodies.

The invention also provides a method of producing a cell populationcomprising selecting from a plurality of cells placental stem cells that(a) adhere to a substrate, (b) express CD200 and HLA-G, and (c)detectably suppress CD4⁺ or CD8⁺ T cell proliferation in an MLR (mixedlymphocyte reaction); and isolating said placental stem cells from othercells to form a cell population. The invention also provides a method ofproducing a cell population comprising selecting from a plurality ofcells placental stem cells that (a) adhere to a substrate, (b) expressCD73, CD105, and CD200, and (c) detectably suppress CD4⁺ or CD8⁺ T cellproliferation in an MLR; and isolating said placental stem cells fromother cells to form a cell population. The invention also provides amethod of producing a cell population comprising selecting placentalstem cells that (a) adhere to a substrate, (b) express CD200 and OCT-4,and (c) detectably suppress CD4⁺ or CD8⁺ T cell proliferation in an MLR;and isolating said placental stem cells from other cells to form a cellpopulation. The invention also provides a method of producing a cellpopulation comprising selecting from a plurality of cells placental stemcells that (a) adhere to a substrate, (b) express CD73 and CD105, (c)form embryoid-like bodies when cultured under conditions allowing theformation of embryoid-like bodies, and (d) detectably suppress CD4⁺ orCD8⁺ T cell proliferation in an MLR; and isolating said placental stemcells from other cells to form a cell population. The invention alsoprovides a method of producing a cell population comprising selectingfrom a plurality of cells placental cells that (a) adhere to asubstrate, (b) express CD73, CD105, and HLA-G, and (c) detectablysuppress CD4⁺ or CD8⁺ T cell proliferation in an MLR; and isolating saidplacental cells from other cells to form a cell population. Theinvention also provides a method of producing a cell populationcomprising selecting from a plurality of cells placental cells that (a)adhere to a substrate, (b) express OCT-4, (c) form embryoid-like bodieswhen cultured under conditions allowing the formation of embryoid-likebodies, and (d) detectably suppress CD4⁺ or CD8⁺ T cell proliferation inan MLR; and isolating said placental cells from other cells to form acell population.

In specific embodiments of any of the embodiments herein, said T cellsand said placental stem cells are present in said MLR at a ratio of,e.g., about 20:1, 15:1, 10:1, 5:1, 2:2, 1:1, 1:2, 1:5, 1:10 or 1:20,preferably 5:1.

In another specific embodiment, the methods comprise selecting cellsthat express ABC-p. In another specific embodiment, the methods compriseselecting cells exhibiting at least one characteristic specific to amesenchymal stem cell. In a more specific embodiment, saidcharacteristic specific to a mesenchymal stem cell is expression ofCD29, expression of CD44, expression of CD90, or expression of acombination of the foregoing. In another specific embodiment of themethods, said selecting is accomplished using an antibody. In anotherspecific embodiment, said selecting is accomplished using flowcytometry. In another specific embodiment, said selecting isaccomplished using magnetic beads. In another specific embodiment, saidselecting is accomplished by fluorescence-activated cell sorting. Inanother specific embodiment of the above methods, said cell populationis expanded.

Placental stem cells used in the methods herein can be derived from thewhole placenta, or from any part of the placenta. For example, invarious embodiments, said placental stem cells are derived primarily, oronly, from amnion, or amnion and chorion, or are derived from placentalperfusate collected during placental perfusion. In specific embodiments,said placental stem cells suppress CD4⁺ or CD8⁺ T cell proliferation byat least 50%, 70%, 90%, or 95% in an MLR compared to an amount of T cellproliferation in said MLR in the absence of said placental stem cells.In another specific embodiment, said placental stem cells additionallydetectably suppress an activity of natural killer (NK) cells.

The invention further provides isolated cell populations comprisingplacental stem cells produced by any of the methods described herein forselecting immunomodulatory placental cell populations, wherein suchpopulation has been identified as detectably suppressing CD4⁺ or CD8⁺ Tcell proliferation in an MLR. For example, in one embodiment, theinvention provides a cell population comprising isolated placental stemcells, wherein said placental stem cells: (a) adhere to a substrate; (b)express CD200 and HLA-G, or express CD73, CD105, and CD200, or expressCD200 and OCT-4, or express CD73, CD105, and HLA-G, or express CD73 andCD105, and facilitate the formation of one or more embryoid-like bodiesin a population of placental cells that comprise the placental stemcells, when said population is cultured under conditions that allowformation of embryoid-like bodies, or express OCT-4 and facilitate theformation of one or more embryoid-like bodies in a population ofplacental cells that comprise the placental stem cells, when saidpopulation is cultured under conditions that allow formation ofembryoid-like bodies, wherein such population has been identified asdetectably suppressing CD4⁺ or CD8⁺ T cell proliferation in an MLR.

The invention also provides an isolated cell population comprisingplacental stem cells that (a) adhere to a substrate, (b) express CD200and HLA-G, and (c) have been identified as detectably suppressing CD4⁺or CD8⁺ T cell proliferation in an MLR. The invention also provides anisolated cell population comprising placental stem cells that (a) adhereto a substrate, (b) express CD73, CD105, and CD200, and (c) have beenidentified as detectably suppressing CD4⁺ or CD8⁺ T cell proliferationin an MLR. The invention also provides an isolated cell populationcomprising placental stem cells that (a) adhere to a substrate, (b)express CD200 and OCT-4, and (c) have been identified as detectablysuppressing CD4⁺ or CD8⁺ T cell proliferation in an MLR. The inventionalso provides an isolated cell population comprising placental stemcells that (a) adhere to a substrate, (b) express CD73 and CD105, (c)form embryoid-like bodies when cultured under conditions allowing theformation of embryoid-like bodies, and (d) have been identified asdetectably suppressing CD4⁺ or CD8⁺ T cell proliferation in an MLR. Theinvention also provides an isolated cell population comprising placentalstem cells that (a) adhere to a substrate, (b) express CD73, CD105, andHLA-G, and (c) have been identified as detectably suppressing CD4⁺ orCD8⁺ T cell proliferation in an MLR. The invention also provides anisolated cell population comprising placental stem cells that (a) adhereto a substrate, (b) express OCT-4, (c) form embryoid-like bodies whencultured under conditions allowing the formation of embryoid-likebodies, and (d) have been identified as detectably suppressing CD4⁺ orCD8⁺ T cell proliferation in an MLR.

In a more specific embodiment of the composition, said placental stemcells that express CD200 and HLA-G also express CD73 and CD105, that is,are CD73⁺ and CD105⁺. In another specific embodiment, said placentalcells are CD34⁻, CD38⁻ or CD45⁻. In a more specific embodiment, saidplacental stem cells are CD34⁻, CD38⁻, CD45⁻, CD73⁺ and CD105⁺. Inanother specific embodiment, said plurality of placental stem cellsfacilitates the development of one or more embryoid-like bodies from apopulation of isolated placental cells comprising the placental stemcells when said population is cultured under conditions that allowformation of embryoid-like bodies.

In another more specific embodiment of the composition, said placentalstem cells that express CD73, CD105, and CD200 are also HLA-G⁺. Inanother specific embodiment, said placental stem cells are CD34⁻, CD38⁻or CD45⁻. In another specific embodiment, said placental stem cells areCD34⁻, CD38⁻ and CD45⁻. In a more specific embodiment, said placentalstem cells are CD34⁻, CD38⁻, CD45⁻, and HLA-G⁺. In another specificembodiment, said placental stem cells facilitate the development of oneor more embryoid-like bodies from a population of isolated placentalcells comprising the placental stem cells when said population iscultured under conditions that allow formation of embryoid-like bodies.

In another more specific embodiment of the composition, said placentalstem cells that express CD200 and OCT-4 also express CD73⁺ and CD105⁺.In another specific embodiment, said placental stem cells are HLA-G⁺. Inanother specific embodiment, said placental stem cells are CD34⁻, CD38⁻or CD45⁻. In another specific embodiment, said placental stem cells areCD34⁻, CD38⁻ and CD45⁻. In a more specific embodiment, said placentalstem cells are CD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺ and HLA-G⁺. In anotherspecific embodiment, said placental stem cells facilitate the formationof one or more embryoid-like bodies from a population of isolatedplacental cells comprising placental stem cells when said population iscultured under conditions that allow formation of embryoid-like bodies.

In another more specific embodiment of the composition, said placentalstem cells that express CD73, CD105, and HLA-G are also CD34, CD38⁻ orCD45⁻. In another specific embodiment, said placental stem cells areCD34⁻, CD38⁻ and CD45⁻. In another specific embodiment, said placentalstem cells are OCT-4⁺. In another specific embodiment, said placentalstem cells are CD200⁺. In a more specific embodiment, said placentalstem cells are CD34⁻, CD45⁻, OCT-4⁺ and CD200⁺. In another specificembodiment, said stem cells facilitate the formation of one or moreembryoid-like bodies from a population of isolated placental cellscomprising the placental stem cells when said population is culturedunder conditions that allow formation of embryoid-like bodies.

In another more specific embodiment of the composition, said placentalstem cells that express CD73 and CD105, and facilitate the formation ofone or more embryoid-like bodies in a population of placental cells thatcomprise the placental stem cells when said population is cultured underconditions that allow formation of embryoid-like bodies, are also CD34⁻,CD38⁻ or CD45⁻. In another specific embodiment, said placental stemcells are OCT-4⁺. In another specific embodiment, said placental stemcells are CD200⁺. In another specific embodiment, said placental stemcells are OCT-4⁺, CD200⁺, CD34⁻, CD38⁻ and CD45⁻.

In another more specific embodiment of the composition, said placentalstem cells that express OCT-4, and facilitate the formation of one ormore embryoid-like bodies in a population of placental cells thatcomprise the placental stem cells when said population is cultured underconditions that allow formation of embryoid-like bodies, are also CD73⁺and CD105⁺. In another specific embodiment, said placental stem cellsare CD34⁻, CD38⁻ and CD45⁻. In another specific embodiment, saidplacental stem cells are CD200⁺. In another specific embodiment, saidplacental stem cells are CD73⁺, CD105⁺, CD200⁺, CD34⁻, CD38⁻ and CD45⁻.

The invention further provides immunomodulatory compositions. In oneembodiment, the invention provides a composition comprising supernatantfrom a culture of any of the cell populations described herein. Inanother embodiment, the invention provides a composition comprisingculture medium from a culture of placental stem cells, wherein saidplacental cells (a) adhere to a substrate; (b) express CD200 and HLA-G,or express CD73, CD105, and CD200, or express CD200 and OCT-4, orexpress CD73, CD105, and HLA-G, or express CD73 and CD105, andfacilitate the formation of one or more embryoid-like bodies in apopulation of placental cells that comprise the placental stem cells,when said population is cultured under conditions that allow formationof embryoid-like bodies, or express OCT-4 and facilitate the formationof one or more embryoid-like bodies in a population of placental cellsthat comprise the placental stem cells, when said population is culturedunder conditions that allow formation of embryoid-like bodies; and (c)detectably suppress CD4⁺ or CD8⁺ T cell proliferation in an MLR (mixedlymphocyte reaction), wherein said culture of placental stem cells havebeen cultured in said medium for 24 hours or more. In a specificembodiment, said composition comprises a plurality of said placentalstem cells. In another specific embodiment, said composition comprises aplurality of non-placental cells. In a more specific embodiment, saidnon-placental cells comprise CD34⁺ cells. The CD34⁺ cells can be, e.g.,peripheral blood hematopoietic progenitor cells, cord bloodhematopoietic progenitor cells, or placental blood hematopoieticprogenitor cells. In another specific embodiment, said non-placentalcells comprise mesenchymal stem cells. In a more specific embodiment,said mesenchymal stem cells are bone marrow-derived mesenchymal stemcells. In another specific embodiment, the composition further comprisesan anti-MIP-1α or anti-MIP-1β antibody.

In another specific embodiment, any of the foregoing compositionscomprises a matrix. In a more specific embodiment, said matrix is athree-dimensional scaffold. In another more specific embodiment, saidmatrix comprises collagen, gelatin, laminin, fibronectin, pectin,ornithine, or vitronectin. In another more specific embodiment, thematrix is an amniotic membrane or an amniotic membrane-derivedbiomaterial. In another more specific embodiment, said matrix comprisesan extracellular membrane protein. In another more specific embodiment,said matrix comprises a synthetic compound. In another more specificembodiment, said matrix comprises a bioactive compound. In another morespecific embodiment, said bioactive compound is a growth factor,cytokine, antibody, or organic molecule of less than 5,000 daltons.

The invention further provides cryopreserved stem cell populations,e.g., a cell population comprising placental stem cells, wherein thecell population is immunomodulatory, that are described herein. Forexample, the invention provides a population of CD200⁺, HLA-G⁺ placentalstem cells that have been identified as detectably suppressing T cellproliferation in a mixed lymphocyte reaction (MLR) assay, wherein saidcells have been cryopreserved, and wherein said population is containedwithin a container. The invention also provides a population of CD73⁺,CD105⁺, CD200⁺ placental stem cells that have been identified asdetectably suppressing T cell proliferation in a mixed lymphocytereaction (MLR) assay, wherein said stem cells have been cryopreserved,and wherein said population is contained within a container. Theinvention also provides a population of CD200⁺, OCT-4⁺ placental stemcells that have been identified as detectably suppressing T cellproliferation in a mixed lymphocyte reaction (MLR) assay, wherein saidstem cells have been cryopreserved, and wherein said population iscontained within a container. The invention also provides a populationof CD73⁺, CD105⁺ placental stem cells that have been identified asdetectably suppressing T cell proliferation in a mixed lymphocytereaction (MLR) assay, wherein said cells have been cryopreserved, andwherein said population is contained within a container, and whereinsaid stem cells facilitate the formation of one or more embryoid-likebodies when cultured with a population of placental cells underconditions that allow for the formation of embryoid-like bodies. Theinvention further provides a population of CD73⁺, CD105⁺, HLA-G⁺placental stem cells that have been identified as detectably suppressingT cell proliferation in a mixed lymphocyte reaction (MLR) assay, whereinsaid cells have been cryopreserved, and wherein said population iscontained within a container. The invention also provides a populationof OCT-4⁺ placental stem cells that have been identified as detectablysuppressing T cell proliferation in a mixed lymphocyte reaction (MLR)assay, wherein said cells have been cryopreserved, wherein saidpopulation is contained within a container, and wherein said stem cellsfacilitate the formation of one or more embryoid-like bodies whencultured with a population of placental cells under conditions thatallow for the formation of embryoid-like bodies.

In a specific embodiment of any of the foregoing cryopreservedpopulations, said container is a bag. In various specific embodiments,said population comprises about, at least, or at most 1×10⁶ said stemcells, 5×10⁶ said stem cells, 1×10⁷ said stem cells, 5×10⁷ said stemcells, 1×10⁸ said stem cells, 5×10⁸ said stem cells, 1×10⁹ said stemcells, 5×10⁹ said stem cells, or 1×10¹⁰ said stem cells. In otherspecific embodiments of any of the foregoing cryopreserved populations,said stem cells have been passaged about, at least, or no more than 5times, no more than 10 times, no more than 15 times, or no more than 20times. In another specific embodiment of any of the foregoingcryopreserved populations, said stem cells have been expanded withinsaid container.

3.1 Definitions

As used herein, the term “SH2” refers to an antibody that binds anepitope on the marker CD105. Thus, cells that are referred to as SH2⁺are CD105⁺.

As used herein, the terms “SH3” and SH4″ refer to antibodies that bindepitopes present on the marker CD73. Thus, cells that are referred to asSH3⁺ and/or SH4⁺ are CD73⁺.

As used herein, the term “isolated stem cell” means a stem cell that issubstantially separated from other, non-stem cells of the tissue, e.g.,placenta, from which the stem cell is derived. A stem cell is “isolated”if at least 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of thenon-stem cells with which the stem cell is naturally associated areremoved from the stem cell, e.g., during collection and/or culture ofthe stem cell.

As used herein, the term “isolated population of cells” means apopulation of cells that is substantially separated from other cells ofthe tissue, e.g., placenta, from which the population of cells isderived. A population of, e.g., stem cells is “isolated” if at least50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of the cells with whichthe population of stem cells are naturally associated are removed fromthe population of stem cells, e.g., during collection and/or culture ofthe population of stem cells.

As used herein, the term “placental stem cell” refers to a stem cell orprogenitor cell that is derived from a mammalian placenta, regardless ofmorphology, cell surface markers, or the number of passages after aprimary culture, which adheres to a tissue culture substrate (e.g.,tissue culture plastic or a fibronectin-coated tissue culture plate).The term “placenta stem cell” as used herein does not, however, refer toa trophoblast. A cell is considered a “stem cell” if the cell retains atleast one attribute of a stem cell, e.g., the ability to differentiateinto at least one other type of cell, or the like.

As used herein, a stem cell is “positive” for a particular marker whenthat marker is detectable. For example, a placental stem cell ispositive for, e.g., CD73 because CD73 is detectable on placental stemcells in an amount detectably greater than background (in comparison to,e.g., an isotype control). A cell is also positive for a marker whenthat marker can be used to distinguish the cell from at least one othercell type, or can be used to select or isolate the cell when present orexpressed by the cell.

As used herein, “immunomodulation” and “immunomodulatory” mean causing,or having the capacity to cause, a detectable change in an immuneresponse, and the ability to cause a detectable change in an immuneresponse.

As used herein, “immunosuppression” and “immunosuppressive” meancausing, or having the capacity to cause, a detectable reduction in animmune response, and the ability to cause a detectable suppression of animmune response.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Viability of placental stem cells from perfusion (A), amnion(B), chorion (C), or amnion-chorion plate (D), or umbilical cord stemcells (E). Numbers on X-axis designate placenta from which stem cellswere obtained.

FIG. 2: Percent HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ cells from perfusion (A),amnion (B), chorion (C), or amnion-chorion plate (D), or umbilical cordstem cells (E) as determined by FACSCalibur. Numbers on X-axis designateplacenta from which stem cells were obtained.

FIG. 3: Percent HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ cells from perfusion (A),amnion (B), chorion (C), or amnion-chorion plate (D), or umbilical cordstem cells (E), as determined by FACS Aria. Numbers on X-axis designateplacenta from which stem cells were obtained.

FIG. 4: HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200expression in stem cells derived from placental perfusate.

FIG. 5: HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200expression in stem cells derived from amnion.

FIG. 6: HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200expression in stem cells derived from chorion.

FIG. 7: HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200expression in stem cells derived from amnion-chorion plate.

FIG. 8: HLA-G, CD10, CD13, CD33, CD38, CD44, CD90, CD105, CD117, CD200expression in stem cells derived from umbilical cord.

FIG. 9: Average expression of HLA-G, CD10, CD13, CD33, CD38, CD44, CD90,CD105, CD117, CD200 expression in stem cells derived from perfusion (A),amnion (B), chorion (C), amnion-chorion plate (D) or umbilical cord (E).

FIGS. 10A and 10B: The mixed lymphocyte reaction (MLR) is a model forthe naïve immune response, and is inhibited by placental stem cells.From the gated “live” and CD8⁺ and CD4⁺ T cell gates, the percentage ofcarboxyfluoroscein succinimidyl ester (CFSE)^(LOW) cells was monitored(FIGS. 10A and 10B, respectively). This percentage increased after a sixday MLR ((FIGS. 10C and 10D, MLR trace), and with the addition ofplacental stem cells (FIGS. 3C and 3D, PMLR trace), the effect isreversed both in the CD8⁺ and CD4⁺ T cell compartments.

FIG. 11: Placenta-derived stem cells from amnion chorionic plate (AC)and umbilical cord stroma (UC) suppress the allo-MLR. The MLR isperformed with either CD4⁺ T cells, CD8⁺ T cells, or equal amounts ofCD4⁺ and CD8⁺ T cells. Abscissa: percent suppression of proliferation.

FIG. 12: Placental stem cells and umbilical cord stem cells inhibit theallo-MLR. A six day assay in round bottom 96 well plate wells. Placentalcells:T cells:Dendritic cells=approximately 1:10:1. Stem cells wereobtained from amnion-chorion (AC), amniotic membrane (AM) or umbilicalcord (UC). FB=fibroblast. BM=bone marrow-derived mesenchymal stem cells.

FIG. 13: Placental stem cells from different donors suppress theallo-MLR to different extents. The figure compares suppression in an MLRby placental stem cells from two placental donors, designated 61665 and63450. Stem cells from placenta 63450 appears to suppress the MLR to agreater degree than stem cells from placenta 61665.

FIG. 14: Seventeen day regression assay and the modified placental stemcell regression assay. The x axis represents the number of placentalstem cells added to the assay. The number of surviving CD23⁺ LCL(lymphoblastoid cell line, an artificially-created transformed B cellline) is measured on the Y axis.

FIG. 15: Placental stem cell suppression of T cell proliferation in thesix day regression assay. A regression assay was set up using CFSEstained T cells. After six days, T cell proliferation was assessed.Relative suppression of T cell proliferation by stem cells fromamnion-chorion (AC), umbilical cord (UC), amniotic membrane (AM), orbone marrow (BM) is shown.

FIG. 16: Percentage change in suppression on introduction of transwellinsert in the MLR, separating placental cells from T cells but allowingexchange of culture medium. Umbilical cord stem cells at 25,000, 50,000,75,000 or 100,000 per reaction show both a relatively high degree ofsuppression and a relatively high degree of need for cell to cellcontact in the high titers to accomplish the suppression.

FIG. 17: Umbilical cord stroma stem cells (UC) added at 12,500 (UC OP/TW12.5) to 100,000 (UC OP/TW 100) were either separated from the NLR by amembrane (TW) or in contact with the MLR (OP). Equal numbers of CD4⁺ Tcells and CD8⁺ T cells were used, and the percentage suppression of theMLR (% CFSE^(Low)=89%) was calculated.

FIG. 18: The relationship between placental stem cell dose and cell tocell contact dependency is not linear. The changes in MLR suppression onintroduction of the insert are calculated from the values given in FIG.17.

FIG. 19: Differential suppression of T cell responses by placental stemcells and BMSCs. The degree of suppression conferred by PDACs or BMSCswas calculated comparing the percentage of MLR T cells in the CFSE^(Lo)gate, more than 70%, to that of the adherent cell MLRs. The MLR waseither separated from the adherent cells (transwell), or was performedin an open well (open). The X axis gives the numbers of adherent cells,in thousands, added to 500,000 T cells and 50,000 DCs. The ratio ofadherent cells to T cells goes from 1:5 to 1:40.

FIG. 20: Differential cell to cell contact requirements for placentalstem cell and bone marrow-derived stem cell immune suppression. From thesuppression data given in FIG. 15, the contact dependency was calculatedand displayed against the adherent cell IT cell ratio (n=3, except UC:n=2).

FIG. 21: T regulatory cells are not required for PDAC T cellsuppression. A regression assay was performed using either whole PBMCs(red and blue graphs) or PBMCs depleted of T regulatory cells (greengraph), both CFSE stained, adding UC PDACs to some conditions (blue andgreen graphs). N=1.

FIG. 22: CFSE^(Hi) cells proliferate in a secondary MLR. From an PDACMLR using CFSE stained cells, the CFSE^(Hi) T cells were isolated on aFACS Aria. The cells were used in an MLR. N=1.

FIG. 23: Supernatant from suppressed stem cell MLR does not suppress MLRat 75% replacement. UC (PUC), AC (PAC), and BMSC (PBM) MLRs wereperformed, and all suppressed the MLR more than 50%. Supernatant fromthe experiments were used to replace from 10 to 150 μl of the 200 μlmedium used for a fresh MLR. As controls, medium from T cell and AC(T/AC) or T cell and bone marrow-derived stem cell (T/BM) cocultureswere also used in the same way (N=2).

FIGS. 24A, 24B: Pre-incubating T cells and adherent cells does notinfluence MLR suppression. T cells from 2 donors were used in twoindependent experiments. Mature DCs (A) or CFSE stained CD3⁺ T cells (B)were incubated with umbilical cord stem cells (UC) or bonemarrow-derived stem cells for the indicated number of days before addingDCs (on day 0, A) or CFSE⁺ CD3⁺ T cells (B, thereby starting the MLR.The adherent cell MLRs then proceeded for six days, as normal. N=2.

FIGS. 25A, 25B: A. MIP-1α and MIP-1β secretion in the MLR, and MLR withplacental stem cells or bone marrow-derived stem cells, correlatesinversely with MLR suppression. B: T cell and NK cell CFSE data from thesame experiment. Supernatants were harvested from the MLR shown in FIG.14B, and analyzed for MIP-1α and MIP-1β. B: MLR was performed asdescribed, and on average 55% (T cells) or 83% (NK cells) CFSE^(Lo)cells were observed. The suppressive effect of stem cell addition wascalculated. N=2 (NK part: N=1).

FIG. 26: In the modified regression assay and MLR supernatants, MCP-1was measured. The placental stem cell suppression of the MLR andregression assay correlates with secretion of the chemoattractant MCP-1.AC: stem cells from amnion-chorion plate. UC: stem cells from umbilicalcord. Light bars: MLR assay results. Dark bars: regression assayresults. Y axis: pg of MCP-1 in assay solution.

FIG. 27: IL-6 measurement in the supernatant of the modified MLR andregression assay. The placental stem cell suppression of the MLR andregression assay correlates with IL-6 secretion. AC: stem cells fromamnion-chorion plate. UC: stem cells from umbilical cord. Light bars:MLR assay results. Dark bars: regression assay results. Y axis: pg ofIL-6 in assay solution.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1 Immunomodulation UsingPlacental Stem Cells

The present invention provides for the modulation, e.g., suppression, ofthe activity, e.g., proliferation, of an immune cell, or plurality ofimmune cells, by contacting the immune cell(s) with a plurality ofplacental stem cells. In one embodiment, the invention provides a methodof suppressing an immune response comprising contacting a plurality ofimmune cells with a plurality of placental stem cells for a timesufficient for said placental stem cells to detectably suppress animmune response, wherein said placental stem cells detectably suppress Tcell proliferation in a mixed lymphocyte reaction (MLR) assay.

Placental stem cells are, e.g., the placental stem cells describedelsewhere herein (see Section 5.2). Placental stem cells used forimmunosuppression can be derived or obtained from a single placenta ormultiple placentas. Placental stem cells used for immunosuppression canalso be derived from a single species, e.g., the species of the intendedrecipient or the species of the immune cells the function of which is tobe reduced or suppressed, or can be derived from multiple species.

An “immune cell” in the context of this method means any cell of theimmune system, particularly T cells and NK (natural killer) cells. Thus,in various embodiments of the method, placental stem cells are contactedwith a plurality of immune cells, wherein the plurality of immune cellsare, or comprises, a plurality of T cells (e.g., a plurality of CD3⁺ Tcells, CD4⁺ T cells and/or CD8⁺ T cells) and/or natural killer cells. An“immune response” in the context of the method can be any response by animmune cell to a stimulus normally perceived by an immune cell, e.g., aresponse to the presence of an antigen. In various embodiments, animmune response can be the proliferation of T cells (e.g., CD3⁺ T cells,CD4⁺ T cells and/or CD8⁺ T cells) in response to a foreign antigen, suchas an antigen present in a transfusion or graft, or to a self-antigen,as in an autoimmune disease. The immune response can also be aproliferation of T cells contained within a graft. The immune responsecan also be any activity of a natural killer (NK) cell, the maturationof a dendritic cell, or the like. The immune response can also be alocal, tissue- or organ-specific, or systemic effect of an activity ofone or more classes of immune cells, e.g. the immune response can begraft versus host disease, inflammation, formation ofinflammation-related scar tissue, an autoimmune condition (e.g.,rheumatoid arthritis, Type I diabetes, lupus erythematosus, etc.). andthe like.

“Contacting” in this context encompasses bringing the placental stemcells and immune cells together in a single container (e.g., culturedish, flask, vial, etc.) or in vivo, for example, the same individual(e.g., mammal, for example, human). In a preferred embodiment, thecontacting is for a time sufficient, and with a sufficient number ofplacental stem cells and immune cells, that a change in an immunefunction of the immune cells is detectable. More preferably, in variousembodiments, said contacting is sufficient to suppress immune function(e.g., T cell proliferation in response to an antigen) by at least 50%,60%, 70%, 80%, 90% or 95%, compared to the immune function in theabsence of the placental stem cells. Such suppression in an in vivocontext can be determined in an in vitro assay (see below); that is, thedegree of suppression in the in vitro assay can be extrapolated, for aparticular number of placental stem cells and a number of immune cellsin a recipient individual, to a degree of suppression in the individual.

The invention in certain embodiments provides methods of using placentalstem cells to modulate an immune response, or the activity of aplurality of one or more types of immune cells, in vitro. Contacting theplacental stem cells and plurality of immune cells can comprisecombining the placental stem cells and immune cells in the same physicalspace such that at least a portion of the plurality of placental stemcells interacts with at least a portion of the plurality of immunecells; maintaining the placental stem cells and immune cells in separatephysical spaces with common medium; or can comprise contacting mediumfrom one or a culture of placental stem cells or immune cells with theother type of cell (for example, obtaining culture medium from a cultureof placental stem cells and resuspending isolated immune cells in themedium). In a specific example, the contacting is a Mixed LymphocyteReaction (MLR).

Such contacting can, for example, take place in an experimental settingdesigned to determine the extent to which a particular plurality ofplacental stem cells is immunomodulatory, e.g., immunosuppressive. Suchan experimental setting can be, for example, a mixed lymphocyte reaction(MLR) or regression assay. Procedures for performing the MLR andregression assays are well-known in the art. See, e.g. Schwarz, “TheMixed Lymphocyte Reaction: An In Vitro Test for Tolerance,” J. Exp. Med.127(5):879-890 (1968); Lacerda et al., “Human Epstein-Barr Virus(EBV)-Specific Cytotoxic T Lymphocytes Home Preferentially to and InduceSelective Regressions of Autologous EBV-Induced B Lymphoproliferationsin Xenografted C.B-17 Scid/Scid Mice,” J. Exp. Med. 183:1215-1228(1996). In a preferred embodiment, an MLR is performed in which aplurality of placental stem cells are contacted with a plurality ofimmune cells (e.g., lymphocytes, for example, CD3⁺, CD4⁺ and/or CD8⁺ Tlymphocytes).

The MLR can be used to determine the immunosuppressive capacity of aplurality of placental stem cells. For example, a plurality of placentalstem cells can be tested in an MLR comprising combining CD4⁺ or CD8⁺ Tcells, dendritic cells (DC) and placental stem cells in a ratio of about10:1:2, wherein the T cells are stained with a dye such as, e.g., CFSEthat partitions into daughter cells, and wherein the T cells are allowedto proliferate for about 6 days. The plurality of placental stem cellsis immunosuppressive if the T cell proliferation at 6 days in thepresence of placental stem cells is detectably reduced compared to Tcell proliferation in the presence of DC and absence of placental stemcells. In such an MLR, placental stem cells are either thawed orharvested from culture. About 20,000 placental stem cells areresuspended in 100 μl of medium (RPMI 1640, 1 mM HEPES buffer,antibiotics, and 5% pooled human serum), and allowed to attach to thebottom of a well for 2 hours. CD4⁺ and/or CD8⁺ T cells are isolated fromwhole peripheral blood mononuclear cells Miltenyi magnetic beads. Thecells are CFSE stained, and a total of 100,000 T cells (CD4⁺ T cellsalone, CD8⁺ T cells alone, or equal amounts of CD4⁺ and CD8⁺ T cells)are added per well. The volume in the well is brought to 200 μl, and theMLR is allowed to proceed.

In one embodiment, therefore, the invention provides a method ofsuppressing an immune response comprising contacting a plurality ofimmune cells with a plurality of placental stem cells for a timesufficient for said placental stem cells to detectably suppress T cellproliferation in a mixed lymphocyte reaction (MLR) assay. In oneembodiment, said placental stem cells used in the MLR represent a sampleor aliquot of placental stem cells from a larger population of placentalstem cells.

Populations of placental stem cells obtained from different placentas,or different tissues within the same placenta, can differ in theirability to modulate an activity of an immune cell, e.g., can differ intheir ability to suppress T cell activity or proliferation or NK cellactivity. It is thus desirable to determine, prior to use, the capacityof a particular population of placental stem cells forimmunosuppression. Such a capacity can be determined, for example, bytesting a sample of the placental stem cell population in an MLR orregression assay. In one embodiment, an MLR is performed with thesample, and a degree of immunosuppression in the assay attributable tothe placental stem cells is determined. This degree of immunosuppressioncan then be attributed to the placental stem cell population that wassampled. Thus, the MLR can be used as a method of determining theabsolute and relative ability of a particular population of placentalstem cells to suppress immune function. The parameters of the MLR can bevaried to provide more data or to best determine the capacity of asample of placental stem cells to immunosuppress. For example, becauseimmunosuppression by placental stem cells appears to increase roughly inproportion to the number of placental stem cells present in the assay,the MLR can be performed with, in one embodiment, two or more numbers ofplacental stem cells, e.g., 1×10³, 3×10³, 1×10⁴ and/or 3×10⁴ placentalstem cells per reaction. The number of placental stem cells relative tothe number of T cells in the assay can also be varied. For example,placental stem cells and T cells in the assay can be present in anyratio of, e.g. about 10:1 to about 1:10, preferably about 1:5, though arelatively greater number of placental stem cells or T cells can beused.

The regression assay can be used in similar fashion.

The invention also provides methods of using placental stem cells tomodulate an immune response, or the activity of a plurality of one ormore types of immune cells, in vivo. Placental stem cells and immunecells can be contacted, e.g., in an individual that is a recipient of aplurality of placental stem cells. Where the contacting is performed inan individual, in one embodiment, the contacting is between exogenousplacental stem cells (that is, placental stem cells not derived from theindividual) and a plurality of immune cells endogenous to theindividual. In specific embodiments, the immune cells within theindividual are CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, and/or NKcells.

Such immunosuppression using placental stem cells would be advantageousfor any condition caused or worsened by, or related to, an inappropriateor undesirable immune response. Placental stem cell-mediatedimmunomodulation, e.g., immunosuppression, would, for example, be usefulin the suppression of an inappropriate immune response raised by theindividual's immune system against one or more of its own tissues. Invarious embodiments, therefore, the invention provides a method ofsuppressing an immune response, wherein the immune response is anautoimmune disease, e.g., lupus erythematosus, diabetes, rheumatoidarthritis, or multiple sclerosis.

The contacting of the plurality of placental stem cells with theplurality of one or more types of immune cells can occur in vivo in thecontext of, or as an adjunct to, for example, grafting or transplantingof one or more types of tissues to a recipient individual. Such tissuesmay be, for example, bone marrow or blood; an organ; a specific tissue(e.g., skin graft); composite tissue allograft (i.e., a graft comprisingtwo or more different types of tissues); etc. In this regard, theplacental stem cells can be used to suppress one or more immuneresponses of one or more immune cells contained within the recipientindividual, within the transplanted tissue or graft, or both. Thecontacting can occur before, during and/or after the grafting ortransplanting. For example, placental stem cells can be administered atthe time of the transplant or graft. The placental stem cells can also,or alternatively, be administered prior to the transplanting orgrafting, e.g., about 1, 2, 3, 4, 5, 6 or 7 days prior to thetransplanting or grafting. Placental stem cells can also, oralternatively, be administered to a transplant or graft recipient afterthe transplantation or grafting, for example, about 1, 2, 3, 4, 5, 6 or7 days after the transplanting or grafting. Preferably, the plurality ofplacental stem cells are contacted with the plurality of placental stemcells before any detectable sign or symptom of an immune response,either by the recipient individual or the transplanted tissue or graft,e.g., a detectable sign or symptom of graft-versus-host disease ordetectable inflammation, is detectable.

In another embodiment, the contacting within an individual is primarilybetween exogenous placental stem cells and exogenous progenitor cells orstem cells, e.g., exogenous progenitor cells or stem cells thatdifferentiate into immune cells. For example, individuals undergoingpartial or full immunoablation or myeloablation as an adjunct to cancertherapy can receive placental stem cells in combination with one or moreother types of stem or progenitor cells. For example, the placental stemcells can be combined with a plurality of CD34⁺ cells, e.g., CD34⁺hematopoietic stem cells. Such CD34⁺ cells can be, e.g., CD34⁺ cellsfrom a tissue source such as peripheral blood, umbilical cord blood,placental blood, or bone marrow. The CD34⁺ cells can be isolated fromsuch tissue sources, or the whole tissue source (e.g., units ofumbilical cord blood or bone marrow) or a partially purified preparationfrom the tissue source (e.g., white blood cells from cord blood) can becombined with the placental stem cells. Combinations of placental stemcells and cord blood, or stem cells from cord blood, are described inHariri, U.S. Application Publication No. 2003/0180269.

The placental stem cells are administered to the individual preferablyin a ratio, with respect to the known or expected number of immunecells, e.g., T cells, in the individual, of from about 10:1 to about1:10, preferably about 1:5. However, a plurality of placental stem cellscan be administered to an individual in a ratio of in non-limitingexamples, about 10,000:1, about 1,000:1, about 100:1, about 10:1, about1:1, about 1:10, about 1:100, about 1:1,000 or about 1:10,000.Generally, about 1×10⁵ to about 1×10⁸ placental stem cells per recipientkilogram, preferably about 1×10⁶ to about 1×10⁷ placental stem perrecipient kilogram can be administered to effect immunosuppression. Invarious embodiments, a plurality of placental stem cells administered toan individual or subject comprises at least, about, or no more than,1×10⁵, 3×10⁵, 1×10⁶, 3×10⁶, 1×10⁷, 3×10⁷, 1×10⁸, 3×10⁸, 1×10⁹, 3×10⁹placental stem cells, or more.

The placental stem cells can also be administered with one or moresecond types of stem cells, e.g., mesenchymal stem cells from bonemarrow. Such second stem cells can be administered to an individual withplacental stem cells in a ratio of, e.g., about 1:10 to about 10:1.

To facilitate contacting the placental stem cells and immune cells invivo, the placental stem cells can be administered to the individual byany route sufficient to bring the placental stem cells and immune cellsinto contact with each other. For example, the placental stem cells canbe administered to the individual, e.g., intravenously, intramuscularly,intraperitoneally, or directly into an organ, e.g., pancreas. For invivo administration, the placental stem cells can be formulated as apharmaceutical composition, as described in Section 5.6.1, below.

The method of immunosuppression can additionally comprise the additionof one or more immunosuppressive agents, particularly in the in vivocontext. In one embodiment, the plurality of placental stem cells arecontacted with the plurality of immune cells in vivo in an individual,and a composition comprising an immunosuppressive agent is administeredto the individual. Immunosuppressive agents are well-known in the artand include, e.g., anti-T cell receptor antibodies (monoclonal orpolyclonal, or antibody fragments or derivatives thereof), anti-IL-2receptor antibodies (e.g., Basiliximab (SIMULECT®) or daclizumab(ZENAPAX)®), anti T cell receptor antibodies (e.g., Muromonab-CD3),azathioprine, corticosteroids, cyclosporine, tacrolimus, mycophenolatemofetil, sirolimus, calcineurin inhibitors, and the like. In a specificembodiment, the immumosuppressive agent is a neutralizing antibody tomacrophage inflammatory protein (MIP)-1α or MIP-1β. Preferably, theanti-MIP-1α or MIP-1β antibody is administered in an amount sufficientto cause a detectable reduction in the amount of MIP-1α and/or MIP-1β insaid individual, e.g., at the time of transplanting.

5.2 Placental Stem Cells and Placental Stem Cell Populations

The methods of immunosuppression of the present invention use placentalstem cells, that is, stem cells obtainable from a placenta or partthereof, that (1) adhere to a tissue culture substrate; (2) have thecapacity to differentiate into non-placental cell types; and (3) have,in sufficient numbers, the capacity to detectably suppress an immunefunction, e.g., proliferation of CD4⁺ and/or CD8⁺ stem cells in a mixedlymphocyte reaction assay. Placental stem cells are not derived fromblood, e.g., placental blood or umbilical cord blood. The placental stemcells used in the methods and compositions of the present invention havethe capacity, and are selected for their capacity, to suppress theimmune system of an individual.

Placental stem cells can be either fetal or maternal in origin (that is,can have the genotype of either the mother or fetus). Populations ofplacental stem cells, or populations of cells comprising placental stemcells, can comprise placental stem cells that are solely fetal ormaternal in origin, or can comprise a mixed population of placental stemcells of both fetal and maternal origin. The placental stem cells, andpopulations of cells comprising the placental stem cells, can beidentified and selected by the morphological, marker, and culturecharacteristics discussed below.

5.2.1 Physical and Morphological Characteristics

The placental stem cells used in the present invention, when cultured inprimary cultures or in cell culture, adhere to the tissue culturesubstrate, e.g., tissue culture container surface (e.g., tissue cultureplastic). Placental stem cells in culture assume a generallyfibroblastoid, stellate appearance, with a number of cyotplasmicprocesses extending from the central cell body. The placental stem cellsare, however, morphologically differentiable from fibroblasts culturedunder the same conditions, as the placental stem cells exhibit a greaternumber of such processes than do fibroblasts. Morphologically, placentalstem cells are also differentiable from hematopoietic stem cells, whichgenerally assume a more rounded, or cobblestone, morphology in culture.

5.2.2 Cell Surface, Molecular and Genetic Markers

Placental stem cells, and populations of placental stem cells, useful inthe methods and compositions of the present invention, express aplurality of markers that can be used to identify and/or isolate thestem cells, or populations of cells that comprise the stem cells. Theplacental stem cells, and stem cell populations of the invention (thatis, two or more placental stem cells) include stem cells and stemcell-containing cell populations obtained directly from the placenta, orany part thereof (e.g., amnion, chorion, placental cotyledons, and thelike). Placental stem cell populations also includes populations of(that is, two or more) placental stem cells in culture, and a populationin a container, e.g., a bag. Placental stem cells are not, however,trophoblasts.

Placental stem cells generally express the markers CD73, CD105, CD200,HLA-G, and/or OCT-4, and do not express CD34, CD38, or CD45. Placentalstem cells can also express HLA-ABC (MHC-1) and HLA-DR. These markerscan be used to identify placental stem cells, and to distinguishplacental stem cells from other stem cell types. Because the placentalstem cells can express CD73 and CD105, they can have mesenchymal stemcell-like characteristics. However, because the placental stem cells canexpress CD200 and HLA-G, a fetal-specific marker, they can bedistinguished from mesenchymal stem cells, e.g., bone marrow-derivedmesenchymal stem cells, which express neither CD200 nor HLA-G. In thesame manner, the lack of expression of CD34, CD38 and/or CD45 identifiesthe placental stem cells as non-hematopoietic stem cells.

In one embodiment, the invention provides an isolated cell populationcomprising a plurality of immunosuppressive placental stem cells thatare CD200⁺, HLA-G⁺, wherein said plurality detectably suppresses T cellproliferation in a mixed lymphocyte reaction (MLR) assay. In a specificembodiment of the isolated populations, said stem cells are also CD73⁺and CD105⁺. In another specific embodiment, said stem cells are alsoCD34⁻, CD38⁻ or CD45⁻. In a more specific embodiment, said stem cellsare also CD34⁻, CD38⁻, CD45⁻, CD73⁺ and CD105⁺. In another embodiment,said isolated population produces one or more embryoid-like bodies whencultured under conditions that allow the formation of embryoid-likebodies.

In another embodiment, the invention provides an isolated cellpopulation comprising a plurality of immunosuppressive placental stemcells that are CD73⁺, CD105⁺, CD200⁺, wherein said plurality detectablysuppress T cell proliferation in a mixed lymphocyte reaction (MLR)assay. In a specific embodiment of said populations, said stem cells areHLA-G⁺. In another specific embodiment, said stem cells are CD34⁻, CD38⁻or CD45⁻. In another specific embodiment, said stem cells are CD34⁻,CD38⁻ and CD45⁻. In a more specific embodiment, said stem cells areCD34⁻, CD38⁻, CD45⁻, and HLA-G⁺. In another specific embodiment, saidpopulation of cells produces one or more embryoid-like bodies whencultured under conditions that allow the formation of embryoid-likebodies.

The invention also provides an isolated cell population comprising aplurality of immunosuppressive placental stem cells that are CD200⁺,OCT-4⁺, wherein said plurality detectably suppresses T cellproliferation in a mixed lymphocyte reaction (MLR) assay. In a specificembodiment, said stem cells are CD73⁺ and CD105⁺. In another specificembodiment, said stem cells are HLA-G⁺. In another specific embodiment,said stem cells are CD34⁻, CD38⁻ and CD45⁻. In a more specificembodiment, said stem cells are CD34⁻, CD38⁻, CD45⁻, CD73⁺, CD105⁺ andHLA-G⁺. In another specific embodiment, the population produces one ormore embryoid-like bodies when cultured under conditions that allow theformation of embryoid-like bodies.

The invention also provides an isolated cell population comprising aplurality of immunosuppressive placental stem cells that are CD73⁺,CD105⁺ and HLA-G⁺, wherein said plurality detectably suppresses T cellproliferation in a mixed lymphocyte reaction (MLR) assay. In a specificembodiment of the above plurality, said stem cells are also CD34⁻, CD38⁻or CD45⁻. In another specific embodiment, said stem cells are alsoCD34⁻, CD38⁻ and CD45⁻. In another specific embodiment, said stem cellsare also OCT-4⁺. In another specific embodiment, said stem cells arealso CD200⁺. In a more specific embodiment, said stem cells are alsoCD34⁻, CD38⁻, CD45⁻, OCT-4⁺ and CD200⁺.

The invention also provides an isolated cell population comprising aplurality of immunosuppressive placental stem cells that are CD73⁺,CD105⁺ stem cells, wherein said plurality forms one or moreembryoid-like bodies under conditions that allow formation ofembryoid-like bodies, and wherein said plurality detectably suppresses Tcell proliferation in a mixed lymphocyte reaction (MLR) assay. In aspecific embodiment, said stem cells are also CD34⁻, CD38⁻ or CD45⁻. Inanother specific embodiment, said stem cells are also CD34⁻, CD38⁻ andCD45⁻. In another specific embodiment, said stem cells are also OCT-4⁺.In a more specific embodiment, said stem cells are also OCT-4⁺, CD34⁻,CD38⁻ and CD45⁻.

The invention also provides an isolated cell population comprising aplurality of immunosuppressive placental stem cells that are OCT-4⁺ stemcells, wherein said population forms one or more embryoid-like bodieswhen cultured under conditions that allow the formation of embryoid-likebodies, and wherein said plurality detectably suppresses T cellproliferation in a mixed lymphocyte reaction (MLR) assay. In variousembodiments, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50% at least 60%, at least 70%, at least 80%, at least 90%, or atleast 95% of said isolated placental cells are OCT4⁺ stem cells. In aspecific embodiment of the above populations, said stem cells are CD73⁺and CD105⁺. In another specific embodiment, said stem cells are CD34⁻,CD38⁻, or CD45⁻. In another specific embodiment, said stem cells areCD200⁺. In a more specific embodiment, said stem cells are CD73⁺,CD105⁺, CD200⁺, CD34⁻, CD38⁻, and CD45⁻. In another specific embodiment,said population has been expanded, for example, passaged at least once,at least three times, at least five times, at least 10 times, at least15 times, or at least 20 times.

In another embodiment, the invention provides an isolated cellpopulation comprising a plurality of immunosuppressive placental stemcells that are CD29⁺, CD44⁺, CD73⁺, CD90⁺, CD105⁺, CD200⁺, CD34⁻ andCD133⁻.

In a specific embodiment of the above-mentioned placental stem cells,the placental stem cells constitutively secrete IL-6, IL-8 and monocytechemoattractant protein (MCP-1).

Each of the above-referenced pluralities of placental stem cells cancomprise placental stem cells obtained and isolated directly from amammalian placenta, or placental stem cells that have been cultured andpassaged at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25,30 or more times, or a combination thereof.

The immunosuppressive pluralities of placental stem cells describedabove can comprise about, at least, or no more than, 1×10⁵, 5×10⁵,1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰,1×10¹¹ or more placental stem cells.

5.2.3 Selecting and Producing Placental Stem Cell Populations

In another embodiment, the invention also provides a method of selectinga plurality of immunosuppressive placental stem cells from a pluralityof placental cells, comprising selecting a population of placental cellswherein at least 10%, at least 20%, at least 30%, at least 40%, at least50% at least 60%, at least 70%, at least 80%, at least 90%, or at least95% of said cells are CD200⁺, HLA-G⁺ placental stem cells, and whereinsaid placental stem cells detectably suppresses T cell proliferation ina mixed lymphocyte reaction (MLR) assay. In a specific embodiment, saidselecting comprises selecting stem cells that are also CD73⁺ and CD105⁺.In another specific embodiment, said selecting comprises selecting stemcells that are also CD34⁻, CD38⁻ or CD45⁻. In another specificembodiment, said selecting comprises selecting placental stem cells thatare also CD34⁻, CD38⁻, CD45⁻, CD73⁺ and CD105⁺. In another specificembodiment, said selecting also comprises selecting a plurality ofplacental stem cells that forms one or more embryoid-like bodies whencultured under conditions that allow the formation of embryoid-likebodies.

In another embodiment, the invention also provides a method of selectinga plurality of immunosuppressive placental stem cells from a pluralityof placental cells, comprising selecting a plurality of placental cellswherein at least 10%, at least 20%, at least 30%, at least 40%, at least50% at least 60%, at least 70%, at least 80%, at least 90%, or at least95% of said cells are CD73⁺, CD105⁺, CD200⁺ placental stem cells, andwherein said placental stem cells detectably suppresses T cellproliferation in a mixed lymphocyte reaction (MLR) assay. In a specificembodiment, said selecting comprises selecting stem cells that are alsoHLA-G⁺. In another specific embodiment, said selecting comprisesselecting placental stem cells that are also CD34⁻, CD38⁻ or CD45⁻. Inanother specific embodiment, said selecting comprises selectingplacental stem cells that are also CD34⁻, CD38⁻ and CD45⁻. In anotherspecific embodiment, said selecting comprises selecting placental stemcells that are also CD34⁻, CD38⁻, CD45⁻, and HLA-G⁺. In another specificembodiment, said selecting additionally comprises selecting a populationof placental cells that produces one or more embryoid-like bodies whenthe population is cultured under conditions that allow the formation ofembryoid-like bodies.

In another embodiment, the invention also provides a method of selectinga plurality of immunosuppressive placental stem cells from a pluralityof placental cells, comprising selecting a plurality of placental cellswherein at least 10%, at least 20%, at least 30%, at least 40%, at least50% at least 60%, at least 70%, at least 80%, at least 90%, or at least95% of said cells are CD200⁺, OCT-4⁺ placental stem cells, and whereinsaid placental stem cells detectably suppresses T cell proliferation ina mixed lymphocyte reaction (MLR) assay. In a specific embodiment, saidselecting comprises selecting placental stem cells that are also CD73⁺and CD105⁺. In another specific embodiment, said selecting comprisesselecting placental stem cells that are also HLA-G⁺. In another specificembodiment, said selecting comprises selecting placental stem cells thatare also CD34⁻, CD38⁻ and CD45⁻. In another specific embodiment, saidselecting comprises selecting placental stem cells that are also CD34⁻,CD38⁻, CD45⁻, CD73⁺, CD105⁺ and HLA-G⁺.

In another embodiment, the invention also provides a method of selectinga plurality of immunosuppressive placental stem cells from a pluralityof placental cells, comprising selecting a plurality of placental cellswherein at least 10%, at least 20%, at least 30%, at least 40%, at least50% at least 60%, at least 70%, at least 80%, at least 90%, or at least95% of said cells are CD73⁺, CD105⁺ and HLA-G⁺ placental stem cells, andwherein said placental stem cells detectably suppresses T cellproliferation in a mixed lymphocyte reaction (MLR) assay. In a specificembodiment, said selecting comprises selecting placental stem cells thatare also CD34⁻, CD38⁻ or CD45⁻. In another specific embodiment, saidselecting comprises selecting placental stem cells that are also CD34⁻,CD38⁻ and CD45⁻. In another specific embodiment, said selectingcomprises selecting placental stem cells that are also CD200⁺. Inanother specific embodiment, said selecting comprises selectingplacental stem cells that are also CD34⁻, CD38, CD45⁻, OCT-4⁺ andCD200⁺.

In another embodiment, the invention also provides a method of selectinga plurality of immunosuppressive placental stem cells from a pluralityof placental cells, comprising selecting a plurality of placental cellswherein at least 10%, at least 20%, at least 30%, at least 40%, at least50% at least 60%, at least 70%, at least 80%, at least 90%, or at least95% of said cells are CD73⁺, CD105⁺ placental stem cells, and whereinsaid plurality forms one or more embryoid-like bodies under conditionsthat allow formation of embryoid-like bodies. In a specific embodiment,said selecting comprises selecting placental stem cells that are alsoCD34⁻, CD38⁻ or CD45⁻. In another specific embodiment, said selectingcomprises selecting placental stem cells that are also CD34⁻, CD38⁻ andCD45⁻. In another specific embodiment, said selecting comprisesselecting placental stem cells that are also OCT-4⁺. In a more specificembodiment, said selecting comprises selecting placental stem cells thatare also OCT-4⁺, CD34⁻, CD38⁻ and CD45⁻.

In another embodiment, the invention also provides a method of selectinga plurality of immunosuppressive placental stem cells from a pluralityof placental cells, comprising selecting a plurality of placental cellswherein at least 10%, at least 20%, at least 30%, at least 40%, at least50% at least 60%, at least 70%, at least 80%, at least 90%, or at least95% of said isolated placental cells are OCT4⁺ stem cells, and whereinsaid plurality forms one or more embryoid-like bodies under conditionsthat allow formation of embryoid-like bodies. In a specific embodiment,said selecting comprises selecting placental stem cells that are alsoCD73⁺ and CD105⁺. In another specific embodiment, said selectingcomprises selecting placental stem cells that are also CD34⁻, CD38⁻, orCD45⁻. In another specific embodiment, said selecting comprisesselecting placental stem cells that are also CD200⁺. In a more specificembodiment, said selecting comprises selecting placental stem cells thatare also CD73⁺, CD105⁺, CD200⁺, CD34⁻, CD38⁻, and CD45⁻.

The invention also provides methods of producing immunosuppressivepopulations, or pluralities, of placental stem cells. For example, theinvention provides a method of producing a cell population, comprisingselecting any of the pluralities of placental stem cells describedabove, and isolating the plurality of placental stem cells from othercells, e.g., other placental cells. In a specific embodiment, theinvention provides a method of producing a cell population comprisingselecting placental cells, wherein said placental cells (a) adhere to asubstrate, (b) express CD200 and HLA-G, or express CD73, CD105, andCD200, or express CD200 and OCT-4, or express CD73, CD105, and HLA-G, orexpress CD73 and CD105 and facilitate the formation of one or moreembryoid-like bodies in a population of placental cells that comprisethe stem cell, when said population is cultured under conditions thatallow formation of embryoid-like bodies, or express OCT-4 and facilitatethe formation of one or more embryoid-like bodies in a population ofplacental cells that comprise the stem cell, when said population iscultured under conditions that allow formation of embryoid-like bodies;and (c) detectably suppress CD4⁺ or CD8⁺ T cell proliferation in an MLR(mixed lymphocyte reaction); and isolating said placental cells fromother cells to form a cell population.

In a more specific embodiment, the invention provides a method ofproducing a cell population comprising selecting placental stem cellsthat (a) adhere to a substrate, (b) express CD200 and HLA-G, and (c)detectably suppress CD4⁺ or CD8⁺ T cell proliferation in an MLR (mixedlymphocyte reaction); and isolating said placental stem cells from othercells to form a cell population. In another specific embodiment, theinvention provides a method of producing a cell population comprisingselecting placental stem cells that (a) adhere to a substrate, (b)express CD73, CD105, and CD200, and (c) detectably suppress CD4⁺ or CD8⁺T cell proliferation in an MLR; and isolating said placental stem cellsfrom other cells to form a cell population. In another specificembodiment, the invention provides a method of producing a cellpopulation comprising selecting placental stem cells that (a) adhere toa substrate, (b) express CD200 and OCT-4, and (c) detectably suppressCD4⁺ or CD8⁺ T cell proliferation in an MLR; and isolating saidplacental stem cells from other cells to form a cell population. Inanother specific embodiment, the invention provides a method ofproducing a cell population comprising selecting placental stem cellsthat (a) adhere to a substrate, (b) express CD73 and CD105, (c) formembryoid-like bodies when cultured under conditions allowing theformation of embryoid-like bodies, and (d) detectably suppress CD4⁺ orCD8⁺ T cell proliferation in an MLR; and isolating said placental stemcells from other cells to form a cell population. In another specificembodiment, the invention provides a method of producing a cellpopulation comprising selecting placental stem cells that (a) adhere toa substrate, (b) express CD73, CD105, and HLA-G, and (c) detectablysuppress CD4⁺ or CD8⁺ T cell proliferation in an MLR; and isolating saidplacental stem cells from other cells to form a cell population. Amethod of producing a cell population comprising selecting placentalstem cells that (a) adhere to a substrate, (b) express OCT-4, (c) formembryoid-like bodies when cultured under conditions allowing theformation of embryoid-like bodies, and (d) detectably suppress CD4⁺ orCD8⁺ T cell proliferation in an MLR; and isolating said placental stemcells from other cells to form a cell population.

In a specific embodiment of the methods of producing animmunosuppressive placental stem cell population, said T cells and saidplacental cells are present in said MLR at a ratio of about 5:1. Theplacental cells used in the method can be derived from the wholeplacenta, or primarily from amnion, or amnion and chorion. In anotherspecific embodiment, the placental cells suppress CD4⁺ or CD8⁺ T cellproliferation by at least 50%, at least 75%, at least 90%, or at least95% in said MLR compared to an amount of T cell proliferation in saidMLR in the absence of said placental cells. The method can additionallycomprise the selection and/or production of a placental stem cellpopulation capable of immunomodulation, e.g., suppression of theactivity of, other immune cells, e.g., an activity of a natural killer(NK) cell.

5.2.4 Growth in Culture

The growth of the placental stem cells described herein, as for anymammalian cell, depends in part upon the particular medium selected forgrowth. Under optimum conditions, placental stem cells typically doublein number in 3-5 days. During culture, the placental stem cells of theinvention adhere to a substrate in culture, e.g. the surface of a tissueculture container (e.g., tissue culture dish plastic, fibronectin-coatedplastic, and the like) and form a monolayer.

Populations of isolated placental cells that comprise the placental stemcells of the invention, when cultured under appropriate conditions, formembryoid-like bodies, that is, three-dimensional clusters of cells growatop the adherent stem cell layer. Cells within the embryoid-like bodiesexpress markers associated with very early stem cells, e.g., OCT-4,Nanog, SSEA3 and SSEA4. Cells within the embryoid-like bodies aretypically not adherent to the culture substrate, as are the placentalstem cells described herein, but remain attached to the adherent cellsduring culture. Embryoid-like body cells are dependent upon the adherentplacental stem cells for viability, as embryoid-like bodies do not formin the absence of the adherent stem cells. The adherent placental stemcells thus facilitate the growth of one or more embryoid-like bodies ina population of placental cells that comprise the adherent placentalstem cells. Without wishing to be bound by theory, the cells of theembryoid-like bodies are thought to grow on the adherent placental stemcells much as embryonic stem cells grow on a feeder layer of cells.Mesenchymal stem cells, e.g., bone marrow-derived mesenchymal stemcells, do not develop embryoid-like bodies in culture.

5.2.5 Differentiation

The placental stem cells, useful in the methods of the presentinvention, are differentiable into different committed cell lineages.For example, the placental stem cells can be differentiated into cellsof an adipogenic, chondrogenic, neurogenic, or osteogenic lineage. Suchdifferentiation can be accomplished by any method known in the art fordifferentiating, e.g., bone marrow-derived mesenchymal stem cells intosimilar cell lineages.

5.3 Methods of Obtaining Placental Stem Cells

5.3.1 Stem Cell Collection Composition

The present invention further provides methods of collecting andisolating placental stem cells. Generally, stem cells are obtained froma mammalian placenta using a physiologically-acceptable solution, e.g.,a stem cell collection composition. A stem cell collection compositionis described in detail in related U.S. Provisional Application No.60/754,969, entitled “Improved Composition for Collecting and PreservingPlacental Stem Cells and Methods of Using the Composition” filed on Dec.29, 2005.

The stem cell collection composition can comprise anyphysiologically-acceptable solution suitable for the collection and/orculture of stem cells, for example, a saline solution (e.g.,phosphate-buffered saline, Kreb's solution, modified Kreb's solution,Eagle's solution, 0.9% NaCl. etc.), a culture medium (e.g., DMEM,H.DMEM, etc.), and the like.

The stem cell collection composition can comprise one or more componentsthat tend to preserve placental stem cells, that is, prevent theplacental stem cells from dying, or delay the death of the placentalstem cells, reduce the number of placental stem cells in a population ofcells that die, or the like, from the time of collection to the time ofculturing. Such components can be, e.g., an apoptosis inhibitor (e.g., acaspase inhibitor or JNK inhibitor); a vasodilator (e.g., magnesiumsulfate, an antihypertensive drug, atrial natriuretic peptide (ANP),adrenocorticotropin, corticotropin-releasing hormone, sodiumnitroprusside, hydralazine, adenosine triphosphate, adenosine,indomethacin or magnesium sulfate, a phosphodiesterase inhibitor, etc.);a necrosis inhibitor (e.g., 2-(1H-Indol-3-yl)-3-pentylamino-maleimide,pyrrolidine dithiocarbamate, or clonazepam); a TNF-α inhibitor; and/oran oxygen-carrying perfluorocarbon (e.g., perfluorooctyl bromide,perfluorodecyl bromide, etc.).

The stem cell collection composition can comprise one or moretissue-degrading enzymes, e.g., a metalloprotease, a serine protease, aneutral protease, an RNase, or a DNase, or the like. Such enzymesinclude, but are not limited to, collagenases (e.g., collagenase I, II,III or IV, a collagenase from Clostridium histolyticum, etc.); dispase,thermolysin, elastase, trypsin, LIBERASE, hyaluronidase, and the like.

The stem cell collection composition can comprise a bacteriocidally orbacteriostatically effective amount of an antibiotic. In certainnon-limiting embodiments, the antibiotic is a macrolide (e.g.,tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime,cefprozil, cefaclor, cefixime or cefadroxil), a clarithromycin, anerythromycin, a penicillin (e.g., penicillin V) or a quinolone (e.g.,ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, astreptomycin, etc. In a particular embodiment, the antibiotic is activeagainst Gram(+) and/or Gram(−) bacteria, e.g., Pseudomonas aeruginosa,Staphylococcus aureus, and the like.

The stem cell collection composition can also comprise one or more ofthe following compounds: adenosine (about 1 mM to about 50 mM);D-glucose (about 20 mM to about 100 mM); magnesium ions (about 1 mM toabout 50 mM); a macromolecule of molecular weight greater than 20,000daltons, in one embodiment, present in an amount sufficient to maintainendothelial integrity and cellular viability (e.g., a synthetic ornaturally occurring colloid, a polysaccharide such as dextran or apolyethylene glycol present at about 25 g/l to about 100 g/l, or about40 g/l to about 60 g/l); an antioxidant (e.g., butylated hydroxyanisole,butylated hydroxytoluene, glutathione, vitamin C or vitamin E present atabout 25 μM to about 100 μM); a reducing agent (e.g., N-acetylcysteinepresent at about 0.1 mM to about 5 mM); an agent that prevents calciumentry into cells (e.g., verapamil present at about 2 μM to about 25 μM);nitroglycerin (e.g., about 0.05 g/L to about 0.2 g/L); an anticoagulant,in one embodiment, present in an amount sufficient to help preventclotting of residual blood (e.g., heparin or hirudin present at aconcentration of about 1000 units/1 to about 100,000 units/l); or anamiloride containing compound (e.g., amiloride, ethyl isopropylamiloride, hexamethylene amiloride, dimethyl amiloride or isobutylamiloride present at about 1.0 μM to about 5 μM).

5.3.2 Collection and Handling of Placenta

Generally, a human placenta is recovered shortly after its expulsionafter birth. In a preferred embodiment, the placenta is recovered from apatient after informed consent and after a complete medical history ofthe patient is taken and is associated with the placenta. Preferably,the medical history continues after delivery. Such a medical history canbe used to coordinate subsequent use of the placenta or the stem cellsharvested therefrom. For example, human placental stem cells can beused, in light of the medical history, for personalized medicine for theinfant associated with the placenta, or for parents, siblings or otherrelatives of the infant.

Prior to recovery of placental stem cells, the umbilical cord blood andplacental blood are removed. In certain embodiments, after delivery, thecord blood in the placenta is recovered. The placenta can be subjectedto a conventional cord blood recovery process. Typically a needle orcannula is used, with the aid of gravity, to exsanguinate the placenta(see, e.g., Anderson, U.S. Pat. No. 5,372,581; Hessel et al., U.S. Pat.No. 5,415,665). The needle or cannula is usually placed in the umbilicalvein and the placenta can be gently massaged to aid in draining cordblood from the placenta. Such cord blood recovery may be performedcommercially, e.g., LifeBank Inc., Cedar Knolls, N.J., ViaCord, CordBlood Registry and Cryocell. Preferably, the placenta is gravity drainedwithout further manipulation so as to minimize tissue disruption duringcord blood recovery.

Typically, a placenta is transported from the delivery or birthing roomto another location, e.g., a laboratory, for recovery of cord blood andcollection of stem cells by, e.g., perfusion or tissue dissociation. Theplacenta is preferably transported in a sterile, thermally insulatedtransport device (maintaining the temperature of the placenta between20-28° C.), for example, by placing the placenta, with clamped proximalumbilical cord, in a sterile zip-lock plastic bag, which is then placedin an insulated container. In another embodiment, the placenta istransported in a cord blood collection kit substantially as described inpending U.S. patent application Ser. No. 11/230,760, filed Sep. 19,2005. Preferably, the placenta is delivered to the laboratory four totwenty-four hours following delivery. In certain embodiments, theproximal umbilical cord is clamped, preferably within 4-5 cm(centimeter) of the insertion into the placental disc prior to cordblood recovery. In other embodiments, the proximal umbilical cord isclamped after cord blood recovery but prior to further processing of theplacenta.

The placenta, prior to stem cell collection, can be stored under sterileconditions and at either room temperature or at a temperature of 5 to25° C. (centigrade). The placenta may be stored for a period of longerthan forty eight hours, and preferably for a period of four totwenty-four hours prior to perfusing the placenta to remove any residualcord blood. The placenta is preferably stored in an anticoagulantsolution at a temperature of 5 to 25° C. (centigrade). Suitableanticoagulant solutions are well known in the art. For example, asolution of heparin or warfarin sodium can be used. In a preferredembodiment, the anticoagulant solution comprises a solution of heparin(e.g., 1% w/w in 1:1000 solution). The exsanguinated placenta ispreferably stored for no more than 36 hours before placental stem cellsare collected.

The mammalian placenta or a part thereof, once collected and preparedgenerally as above, can be treated in any art-known manner, e.g., can beperfused or disrupted, e.g., digested with one or more tissue-disruptingenzymes, to obtain stem cells.

5.3.3 Physical Disruption and Enzymatic Digestion of Placental Tissue

In one embodiment, stem cells are collected from a mammalian placenta byphysical disruption, e.g., enzymatic digestion, of the organ. Forexample, the placenta, or a portion thereof, may be, e.g., crushed,sheared, minced, diced, chopped, macerated or the like, while in contactwith the stem cell collection composition of the invention, and thetissue subsequently digested with one or more enzymes. The placenta, ora portion thereof, may also be physically disrupted and digested withone or more enzymes, and the resulting material then immersed in, ormixed into, the stem cell collection composition of the invention. Anymethod of physical disruption can be used, provided that the method ofdisruption leaves a plurality, more preferably a majority, and morepreferably at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the cells insaid organ viable, as determined by, e.g., trypan blue exclusion.

The placenta can be dissected into components prior to physicaldisruption and/or enzymatic digestion and stem cell recovery. Forexample, placental stem cells can be obtained from the amnioticmembrane, chorion, placental cotyledons, or any combination thereof.Preferably, placental stem cells are obtained from placental tissuecomprising amnion and chorion. Typically, placental stem cells can beobtained by disruption of a small block of placental tissue, e.g., ablock of placental tissue that is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900 or about 1000 cubic millimeters in volume.

A preferred stem cell collection composition comprises one or moretissue-disruptive enzyme(s). Enzymatic digestion preferably uses acombination of enzymes, e.g., a combination of a matrix metalloproteaseand a neutral protease, for example, a combination of collagenase anddispase. In one embodiment, enzymatic digestion of placental tissue usesa combination of a matrix metalloprotease, a neutral protease, and amucolytic enzyme for digestion of hyaluronic acid, such as a combinationof collagenase, dispase, and hyaluronidase or a combination of LIBERASE(Boehringer Mannheim Corp., Indianapolis, Ind.) and hyaluronidase. Otherenzymes that can be used to disrupt placenta tissue include papain,deoxyribonucleases, serine proteases, such as trypsin, chymotrypsin, orelastase. Serine proteases may be inhibited by alpha 2 microglobulin inserum and therefore the medium used for digestion is usually serum-free.EDTA and DNase are commonly used in enzyme digestion procedures toincrease the efficiency of cell recovery. The digestate is preferablydiluted so as to avoid trapping stem cells within the viscous digest.

Any combination of tissue digestion enzymes can be used. Typicalconcentrations for tissue digestion enzymes include, e.g., 50-200 U/mLfor collagenase I and collagenase IV, 1-10 U/mL for dispase, and 10-100U/mL for elastase. Proteases can be used in combination, that is, two ormore proteases in the same digestion reaction, or can be usedsequentially in order to liberate placental stem cells. For example, inone embodiment, a placenta, or part thereof, is digested first with anappropriate amount of collagenase I at 2 mg/ml for 30 minutes, followedby digestion with trypsin, 0.25%, for 10 minutes, at 37° C. Serineproteases are preferably used consecutively following use of otherenzymes.

In another embodiment, the tissue can further be disrupted by theaddition of a chelator, e.g., ethylene glycol bis(2-aminoethylether)-N,N,N′N′-tetraacetic acid (EGTA) or ethylenediaminetetraaceticacid (EDTA) to the stem cell collection composition comprising the stemcells, or to a solution in which the tissue is disrupted and/or digestedprior to isolation of the stem cells with the stem cell collectioncomposition.

It will be appreciated that where an entire placenta, or portion of aplacenta comprising both fetal and maternal cells (for example, wherethe portion of the placenta comprises the chorion or cotyledons), theplacental stem cells collected will comprise a mix of placental stemcells derived from both fetal and maternal sources. Where a portion ofthe placenta that comprises no, or a negligible number of, maternalcells (for example, amnion), the placental stem cells collected willcomprise almost exclusively fetal placental stem cells.

5.3.4 Placental Perfusion

Placental stem cells can also be obtained by perfusion of the mammalianplacenta. Methods of perfusing mammalian placenta to obtain stem cellsare disclosed, e.g., in Hariri, U.S. Application Publication No.2002/0123141, and in related U.S. Provisional Application No.60/754,969, entitled “Improved Composition for Collecting and PreservingPlacental Stem Cells and Methods of Using the Composition” filed on Dec.29, 2005.

Placental stem cells can be collected by perfusion, e.g., through theplacental vasculature, using, e.g., a stem cell collection compositionas a perfusion solution. In one embodiment, a mammalian placenta isperfused by passage of perfusion solution through either or both of theumbilical artery and umbilical vein. The flow of perfusion solutionthrough the placenta may be accomplished using, e.g., gravity flow intothe placenta. Preferably, the perfusion solution is forced through theplacenta using a pump, e.g., a peristaltic pump. The umbilical vein canbe, e.g., cannulated with a cannula, e.g., a TEFLON® or plastic cannula,that is connected to a sterile connection apparatus, such as steriletubing. The sterile connection apparatus is connected to a perfusionmanifold.

In preparation for perfusion, the placenta is preferably oriented (e.g.,suspended) in such a manner that the umbilical artery and umbilical veinare located at the highest point of the placenta. The placenta can beperfused by passage of a perfusion fluid, e.g., the stem cell collectioncomposition of the invention, through the placental vasculature, orthrough the placental vasculature and surrounding tissue. In oneembodiment, the umbilical artery and the umbilical vein are connectedsimultaneously to a pipette that is connected via a flexible connectorto a reservoir of the perfusion solution. The perfusion solution ispassed into the umbilical vein and artery. The perfusion solution exudesfrom and/or passes through the walls of the blood vessels into thesurrounding tissues of the placenta, and is collected in a suitable openvessel from the surface of the placenta that was attached to the uterusof the mother during gestation. The perfusion solution may also beintroduced through the umbilical cord opening and allowed to flow orpercolate out of openings in the wall of the placenta which interfacedwith the maternal uterine wall. In another embodiment, the perfusionsolution is passed through the umbilical veins and collected from theumbilical artery, or is passed through the umbilical artery andcollected from the umbilical veins.

In one embodiment, the proximal umbilical cord is clamped duringperfusion, and more preferably, is clamped within 4-5 cm (centimeter) ofthe cord's insertion into the placental disc.

The first collection of perfusion fluid from a mammalian placenta duringthe exsanguination process is generally colored with residual red bloodcells of the cord blood and/or placental blood. The perfusion fluidbecomes more colorless as perfusion proceeds and the residual cord bloodcells are washed out of the placenta. Generally from 30 to 100 ml(milliliter) of perfusion fluid is adequate to initially exsanguinatethe placenta, but more or less perfusion fluid may be used depending onthe observed results.

The volume of perfusion liquid used to collect placental stem cells mayvary depending upon the number of stem cells to be collected, the sizeof the placenta, the number of collections to be made from a singleplacenta, etc. In various embodiments, the volume of perfusion liquidmay be from 50 mL to 5000 mL, 50 mL to 4000 mL, 50 mL to 3000 mL, 100 mLto 2000 mL, 250 mL to 2000 mL, 500 mL to 2000 mL, or 750 mL to 2000 mL.Typically, the placenta is perfused with 700-800 mL of perfusion liquidfollowing exsanguination.

The placenta can be perfused a plurality of times over the course ofseveral hours or several days. Where the placenta is to be perfused aplurality of times, it may be maintained or cultured under asepticconditions in a container or other suitable vessel, and perfused withthe stem cell collection composition, or a standard perfusion solution(e.g., a normal saline solution such as phosphate buffered saline(“PBS”)) with or without an anticoagulant (e.g., heparin, warfarinsodium, coumarin, bishydroxycoumarin), and/or with or without anantimicrobial agent (e.g., β-mercaptoethanol (0.1 mM); antibiotics suchas streptomycin (e.g., at 40-100 μg/ml), penicillin (e.g., at 40 U/ml),amphotericin B (e.g., at 0.5 μg/ml). In one embodiment, an isolatedplacenta is maintained or cultured for a period of time withoutcollecting the perfusate, such that the placenta is maintained orcultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, or 24 hours, or 2 or 3 or more days beforeperfusion and collection of perfusate. The perfused placenta can bemaintained for one or more additional time(s), e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 ormore hours, and perfused a second time with, e.g., 700-800 mL perfusionfluid. The placenta can be perfused 1, 2, 3, 4, 5 or more times, forexample, once every 1, 2, 3, 4, 5 or 6 hours. In a preferred embodiment,perfusion of the placenta and collection of perfusion solution, e.g.,stem cell collection composition, is repeated until the number ofrecovered nucleated cells falls below 100 cells/ml. The perfusates atdifferent time points can be further processed individually to recovertime-dependent populations of cells, e.g., stem cells. Perfusates fromdifferent time points can also be pooled.

Without wishing to be bound by any theory, after exsanguination and asufficient time of perfusion of the placenta, placental stem cells arebelieved to migrate into the exsanguinated and perfused microcirculationof the placenta where, according to the methods of the invention, theyare collected, preferably by washing into a collecting vessel byperfusion. Perfusing the isolated placenta not only serves to removeresidual cord blood but also provide the placenta with the appropriatenutrients, including oxygen. The placenta may be cultivated and perfusedwith a similar solution which was used to remove the residual cord bloodcells, preferably, without the addition of anticoagulant agents.

Perfusion according to the methods of the invention results in thecollection of significantly more placental stem cells than the numberobtainable from a mammalian placenta not perfused with said solution,and not otherwise treated to obtain stem cells (e.g., by tissuedisruption, e.g., enzymatic digestion). In this context, “significantlymore” means at least 10% more. Perfusion according to the methods of theinvention yields significantly more placental stem cells than, e.g., thenumber of placental stem cells obtainable from culture medium in which aplacenta, or portion thereof, has been cultured.

Stem cells can be isolated from placenta by perfusion with a solutioncomprising one or more proteases or other tissue-disruptive enzymes. Ina specific embodiment, a placenta or portion thereof (e.g., amnioticmembrane, amnion and chorion, placental lobule or cotyledon, orcombination of any of the foregoing) is brought to 25-37° C., and isincubated with one or more tissue-disruptive enzymes in 200 mL of aculture medium for 30 minutes. Cells from the perfusate are collected,brought to 4° C., and washed with a cold inhibitor mix comprising 5 mMEDTA, 2 mM dithiothreitol and 2 mM beta-mercaptoethanol. The stem cellsare washed after several minutes with a cold (e.g., 4° C.) stem cellcollection composition of the invention.

It will be appreciated that perfusion using the pan method, that is,whereby perfusate is collected after it has exuded from the maternalside of the placenta, results in a mix of fetal and maternal cells. As aresult, the cells collected by this method comprise a mixed populationof placental stem cells of both fetal and maternal origin. In contrast,perfusion solely through the placental vasculature, whereby perfusionfluid is passed through one or two placental vessels and is collectedsolely through the remaining vessel(s), results in the collection of apopulation of placental stem cells almost exclusively of fetal origin.

5.3.5 Isolation, Sorting, and Characterization of Placental Stem Cells

Stem cells from mammalian placenta, whether obtained by perfusion orenyzmatic digestion, can initially be purified from (i.e., be isolatedfrom) other cells by Ficoll gradient centrifugation. Such centrifugationcan follow any standard protocol for centrifugation speed, etc. In oneembodiment, for example, cells collected from the placenta are recoveredfrom perfusate by centrifugation at 5000×g for 15 minutes at roomtemperature, which separates cells from, e.g., contaminating debris andplatelets. In another embodiment, placental perfusate is concentrated toabout 200 ml, gently layered over Ficoll, and centrifuged at about1100×g for 20 minutes at 22° C., and the low-density interface layer ofcells is collected for further processing.

Cell pellets can be resuspended in fresh stem cell collectioncomposition, or a medium suitable for stem cell maintenance, e.g., IMDMserum-free medium containing 2 U/ml heparin and 2 mM EDTA (GibcoBRL,NY). The total mononuclear cell fraction can be isolated, e.g., usingLymphoprep (Nycomed Pharma, Oslo, Norway) according to themanufacturer's recommended procedure.

As used herein, “isolating” placental stem cells means to remove atleast 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the cellswith which the stem cells are normally associated in the intactmammalian placenta. A stem cell from an organ is “isolated” when it ispresent in a population of cells that comprises fewer than 50% of thecells with which the stem cell is normally associated in the intactorgan.

Placental cells obtained by perfusion or digestion can, for example, befurther, or initially, isolated by differential trypsinization using,e.g., a solution of 0.05% trypsin with 0.2% EDTA (Sigma, St. Louis Mo.).Differential trypsinization is possible because placental stem cellstypically detach from plastic surfaces within about five minutes whereasother adherent populations typically require more than 20-30 minutesincubation. The detached placental stem cells can be harvested followingtrypsinization and trypsin neutralization, using, e.g., TrypsinNeutralizing Solution (TNS, Cambrex). In one embodiment of isolation ofadherent cells, aliquots of, for example, about 5-10×10⁶ cells areplaced in each of several T-75 flasks, preferably fibronectin-coated T75flasks. In such an embodiment, the cells can be cultured withcommercially available Mesenchymal Stem Cell Growth Medium (MSCGM)(Cambrex), and placed in a tissue culture incubator (37° C., 5% CO₂).After 10 to 15 days, non-adherent cells are removed from the flasks bywashing with PBS. The PBS is then replaced by MSCGM. Flasks arepreferably examined daily for the presence of various adherent celltypes and in particular, for identification and expansion of clusters offibroblastoid cells.

The number and type of cells collected from a mammalian placenta can bemonitored, for example, by measuring changes in morphology and cellsurface markers using standard cell detection techniques such as flowcytometry, cell sorting, immunocytochemistry (e.g., staining with tissuespecific or cell-marker specific antibodies) fluorescence activated cellsorting (FACS), magnetic activated cell sorting (MACS), by examinationof the morphology of cells using light or confocal microscopy, and/or bymeasuring changes in gene expression using techniques well known in theart, such as PCR and gene expression profiling. These techniques can beused, too, to identify cells that are positive for one or moreparticular markers. For example, using antibodies to CD34, one candetermine, using the techniques above, whether a cell comprises adetectable amount of CD34; if so, the cell is CD34⁺. Likewise, if a cellproduces enough OCT-4 RNA to be detectable by RT-PCR, or significantlymore OCT-4 RNA than an adult cell, the cell is OCT-4⁺ Antibodies to cellsurface markers (e.g., CD markers such as CD34) and the sequence of stemcell-specific genes, such as OCT-4, are well-known in the art.

Placental cells, particularly cells that have been isolated by Ficollseparation, differential adherence, or a combination of both, may besorted using a fluorescence activated cell sorter (FACS). Fluorescenceactivated cell sorting (FACS) is a well-known method for separatingparticles, including cells, based on the fluorescent properties of theparticles (Kamarch, 1987, Methods Enzymol, 151:150-165). Laserexcitation of fluorescent moieties in the individual particles resultsin a small electrical charge allowing electromagnetic separation ofpositive and negative particles from a mixture. In one embodiment, cellsurface marker-specific antibodies or ligands are labeled with distinctfluorescent labels. Cells are processed through the cell sorter,allowing separation of cells based on their ability to bind to theantibodies used. FACS sorted particles may be directly deposited intoindividual wells of 96-well or 384-well plates to facilitate separationand cloning.

In one sorting scheme, stem cells from placenta are sorted on the basisof expression of the markers CD34, CD38, CD44, CD45, CD73, CD105, OCT-4and/or HLA-G. This can be accomplished in connection with procedures toselect stem cells on the basis of their adherence properties in culture.For example, an adherence selection can be accomplished before or aftersorting on the basis of marker expression. In one embodiment, forexample, cells are sorted first on the basis of their expression ofCD34; CD34⁻ cells are retained, and cells that are CD200⁺HLA-G⁺, areseparated from all other CD34⁻ cells. In another embodiment, cells fromplacenta are based on their expression of markers CD200 and/or HLA-G;for example, cells displaying either of these markers are isolated forfurther use. Cells that express, e.g., CD200 and/or HLA-G can, in aspecific embodiment, be further sorted based on their expression of CD73and/or CD105, or epitopes recognized by antibodies SH2, SH3 or SH4, orlack of expression of CD34, CD38 or CD45. For example, in oneembodiment, placental cells are sorted by expression, or lack thereof,of CD200, HLA-G, CD73, CD105, CD34, CD38 and CD45, and placental cellsthat are CD200⁺, HLA-G⁺, CD73⁺, CD105⁺, CD34⁻, CD38⁻ and CD45⁻ areisolated from other placental cells for further use.

In another embodiment, magnetic beads can be used to separate cells. Thecells may be sorted using a magnetic activated cell sorting (MACS)technique, a method for separating particles based on their ability tobind magnetic beads (0.5-100 μm diameter). A variety of usefulmodifications can be performed on the magnetic microspheres, includingcovalent addition of antibody that specifically recognizes a particularcell surface molecule or hapten. The beads are then mixed with the cellsto allow binding. Cells are then passed through a magnetic field toseparate out cells having the specific cell surface marker. In oneembodiment, these cells can then isolated and re-mixed with magneticbeads coupled to an antibody against additional cell surface markers.The cells are again passed through a magnetic field, isolating cellsthat bound both the antibodies. Such cells can then be diluted intoseparate dishes, such as microtiter dishes for clonal isolation.

Placental stem cells can also be characterized and/or sorted based oncell morphology and growth characteristics. For example, placental stemcells can be characterized as having, and/or selected on the basis of,e.g., a fibroblastoid appearance in culture. Placental stem cells canalso be characterized as having, and/or be selected, on the basis oftheir ability to form embryoid-like bodies. In one embodiment, forexample, placental cells that are fibroblastoid in shape, express CD73and CD105, and produce one or more embryoid-like bodies in culture areisolated from other placental cells. In another embodiment, OCT-4⁺placental cells that produce one or more embryoid-like bodies in cultureare isolated from other placental cells.

In another embodiment, placental stem cells can be identified andcharacterized by a colony forming unit assay. Colony forming unit assaysare commonly known in the art, such as Mesen Cult™ medium (Stem CellTechnologies, Inc., Vancouver British Columbia) Placental stem cells canbe assessed for viability, proliferation potential, and longevity usingstandard techniques known in the art, such as trypan blue exclusionassay, fluorescein diacetate uptake assay, propidium iodide uptake assay(to assess viability); and thymidine uptake assay, MTT cellproliferation assay (to assess proliferation). Longevity may bedetermined by methods well known in the art, such as by determining themaximum number of population doubling in an extended culture.

Placental stem cells can also be separated from other placental cellsusing other techniques known in the art, e.g., selective growth ofdesired cells (positive selection), selective destruction of unwantedcells (negative selection); separation based upon differential cellagglutinability in the mixed population as, for example, with soybeanagglutinin; freeze-thaw procedures; filtration; conventional and zonalcentrifugation; centrifugal elutriation (counter-streamingcentrifugation); unit gravity separation; countercurrent distribution;electrophoresis; and the like.

5.4 Culture of Placental Stem Cells

5.4.1 Culture Media

Isolated placental stem cells, or placental stem cell population, orcells or placental tissue from which placental stem cells grow out, canbe used to initiate, or seed, cell cultures. Cells are generallytransferred to sterile tissue culture vessels either uncoated or coatedwith extracellular matrix or ligands such as laminin, collagen (e.g.,native or denatured), gelatin, fibronectin, ornithine, vitronectin, andextracellular membrane protein (e.g., MATRIGEL (BD Discovery Labware,Bedford, Mass.)).

Placental stem cells can be cultured in any medium, and under anyconditions, recognized in the art as acceptable for the culture of stemcells. Preferably, the culture medium comprises serum. Placental stemcells can be cultured in, for example, DMEM-LG (Dulbecco's ModifiedEssential Medium, low glucose)/MCDB 201 (chick fibroblast basal medium)containing ITS (insulin-transferrin-selenium), LA+BSA (linoleicacid-bovine serum albumin), dextrose, L-ascorbic acid, PDGF, EGF, IGF-1,and penicillin/streptomycin; DMEM-HG (high glucose) comprising 10% fetalbovine serum (FBS); DMEM-HG comprising 15% FBS; IMDM (Iscove's modifiedDulbecco's medium) comprising 10% FBS, 10% horse serum, andhydrocortisone; M199 comprising 10% FBS, EGF, and heparin; α-MEM(minimal essential medium) comprising 10% FBS, GlutaMAX™ and gentamicin;DMEM comprising 10% FBS, GlutaMAX™ and gentamicin, etc. A preferredmedium is DMEM-LG/MCDB-201 comprising 2% FBS, ITS, LA+BSA, dextrose,L-ascorbic acid, PDGF, EGF, and penicillin/streptomycin.

Other media in that can be used to culture placental stem cells includeDMEM (high or low glucose), Eagle's basal medium, Ham's F10 medium(F10), Ham's F-12 medium (F12), Iscove's modified Dulbecco's medium,Mesenchymal Stem Cell Growth Medium (MSCGM), Liebovitz's L-15 medium,MCDB, DMIEM/F12, RPMI 1640, advanced DMEM (Gibco), DMEM/MCDB201 (Sigma),and CELL-GRO FREE.

The culture medium can be supplemented with one or more componentsincluding, for example, serum (e.g., fetal bovine serum (FBS),preferably about 2-15% (v/v); equine (horse) serum (ES); human serum(HS)); beta-mercaptoethanol (BME), preferably about 0.001% (v/v); one ormore growth factors, for example, platelet-derived growth factor (PDGF),epidermal growth factor (EGF), basic fibroblast growth factor (bFGF),insulin-like growth factor-1 (IGF-1), leukemia inhibitory factor (LIF),vascular endothelial growth factor (VEGF), and erythropoietin (EPO);amino acids, including L-valine; and one or more antibiotic and/orantimycotic agents to control microbial contamination, such as, forexample, penicillin G, streptomycin sulfate, amphotericin B, gentamicin,and nystatin, either alone or in combination.

5.4.2 Expansion and Proliferation of Placental Stem Cells

Once an isolated placental stem cell, or isolated population of stemcells (e.g., a stem cell or population of stem cells separated from atleast 50% of the placental cells with which the stem cell or populationof stem cells is normally associated in vivo), the stem cell orpopulation of stem cells can be proliferated and expanded in vitro. Forexample, a population of placental stem cells can be cultured in tissueculture containers, e.g., dishes, flasks, multiwell plates, or the like,for a sufficient time for the stem cells to proliferate to 70-90%confluence, that is, until the stem cells and their progeny occupy70-90% of the culturing surface area of the tissue culture container.

Placental stem cells can be seeded in culture vessels at a density thatallows cell growth. For example, the cells may be seeded at low density(e.g., about 1,000 to about 5,000 cells/cm²) to high density (e.g.,about 50,000 or more cells/cm²). In a preferred embodiment, the cellsare cultured at about 0 to about 5 percent by volume CO₂ in air. In somepreferred embodiments, the cells are cultured at about 2 to about 25percent O₂ in air, preferably about 5 to about 20 percent O₂ in air. Thecells preferably are cultured at about 25° C. to about 40° C.,preferably 37° C. The cells are preferably cultured in an incubator. Theculture medium can be static or agitated, for example, using abioreactor. Placental stem cells preferably are grown under lowoxidative stress (e.g., with addition of glutathione, ascorbic acid,catalase, tocopherol, N-acetylcysteine, or the like).

Once 70%-90% confluence is obtained, the cells may be passaged. Forexample, the cells can be enzymatically treated, e.g., trypsinized,using techniques well-known in the art, to separate them from the tissueculture surface. After removing the cells by pipetting and counting thecells, about 20,000-100,000 stem cells, preferably about 50,000 stemcells, are passaged to a new culture container containing fresh culturemedium. Typically, the new medium is the same type of medium from whichthe stem cells were removed. The invention encompasses populations ofplacental stem cells that have been passaged at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 14, 16, 18, or 20 times, or more.

5.4.3 Placental Stem Cell Populations

The invention provides populations of placental stem cells. Placentalstem cell population can be isolated directly from one or moreplacentas; that is, the placental stem cell population can be apopulation of placental cells, comprising placental stem cells, obtainedfrom, or contained within, perfusate, or obtained from, or containedwithin, digestate (that is, the collection of cells obtained byenzymatic digestion of a placenta or part thereof). Isolated placentalstem cells of the invention can also be cultured and expanded to produceplacental stem cell populations. Populations of placental cellscomprising placental stem cells can also be cultured and expanded toproduce placental stem cell populations.

Placental stem cell populations of the invention comprise placental stemcells, for example, placental stem cells as described herein. In variousembodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,or 99% of the cells in an isolated placental stem cell population areplacental stem cells. That is, a placental stem cell population cancomprise, e.g., as much as 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% non-stem cells.

The invention provides methods of producing isolated placental stem cellpopulation by, e.g., selecting placental stem cells, whether derivedfrom enzymatic digestion or perfusion, that express particular markersand/or particular culture or morphological characteristics. In oneembodiment, for example, the invention provides a method of producing acell population comprising selecting placental cells that (a) adhere toa substrate, and (b) express CD200 and HLA-G; and isolating said cellsfrom other cells to form a cell population. In another embodiment, themethod of producing a cell population comprises selecting placentalcells that (a) adhere to a substrate, and (b) express CD73, CD105, andCD200; and isolating said cells from other cells to form a cellpopulation. In another embodiment, the method of producing a cellpopulation comprises selecting placental cells that (a) adhere to asubstrate and (b) express CD200 and OCT-4; and isolating said cells fromother cells to form a cell population. In another embodiment, the methodof producing a cell population comprises selecting placental cells that(a) adhere to a substrate, (b) express CD73 and CD105, and (c)facilitate the formation of one or more embryoid-like bodies in apopulation of placental cells comprising said stem cell when saidpopulation is cultured under conditions that allow for the formation ofan embryoid-like body; and isolating said cells from other cells to forma cell population. In another embodiment, the method of producing a cellpopulation comprises selecting placental cells that (a) adhere to asubstrate, and (b) express CD73, CD105 and HLA-G; and isolating saidcells from other cells to form a cell population. In another embodiment,the method of producing a cell population comprises selecting placentalcells that (a) adhere to a substrate, (b) express OCT-4, and (c)facilitate the formation of one or more embryoid-like bodies in apopulation of placental cells comprising said stem cell when saidpopulation is cultured under conditions that allow for the formation ofan embryoid-like body; and isolating said cells from other cells to forma cell population. In any of the above embodiments, the method canadditionally comprise selecting placental cells that express ABC-p (aplacenta-specific ABC transporter protein; see, e.g., Allikmets et al.,Cancer Res. 58(23):5337-9 (1998)). The method can also compriseselecting cells exhibiting at least one characteristic specific to,e.g., a mesenchymal stem cell, for example, expression of CD29,expression of CD44, expression of CD90, or expression of a combinationof the foregoing.

In the above embodiments, the substrate can be any surface on whichculture and/or selection of cells, e.g., placental stem cells, can beaccomplished. Typically, the substrate is plastic, e.g., tissue culturedish or multiwell plate plastic. Tissue culture plastic can be coatedwith a biomolecule, e.g., laminin or fibronectin.

Cells, e.g., placental stem cells, can be selected for a placental stemcell population by any means known in the art of cell selection. Forexample, cells can be selected using an antibody or antibodies to one ormore cell surface markers, for example, in flow cytometry or FACS.Selection can be accomplished using antibodies in conjunction withmagnetic beads. Antibodies that are specific for certain stemcell-related markers are known in the art. For example, antibodies toOCT-4 (Abeam, Cambridge, Mass.), CD200 (Abcam), HLA-G (Abeam), CD73 (BDBiosciences Pharmingen, San Diego, Calif.), CD105 (Abeam; BioDesignInternational, Saco, Me.), etc. Antibodies to other markers are alsoavailable commercially, e.g., CD34, CD38 and CD45 are available from,e.g., StemCell Technologies or BioDesign International.

The isolated placental stem cell population can comprise placental cellsthat are not stem cells, or cells that are not placental cells.

Isolated placental stem cell populations can be combined with one ormore populations of non-stem cells or non-placental cells. For example,an isolated population of placental stem cells can be combined withblood (e.g., placental blood or umbilical cord blood), blood-derivedstem cells (e.g., stem cells derived from placental blood or umbilicalcord blood), populations of blood-derived nucleated cells, bonemarrow-derived mesenchymal cells, bone-derived stem cell populations,crude bone marrow, adult (somatic) stem cells, populations of stem cellscontained within tissue, cultured stem cells, populations offully-differentiated cells (e.g., chondrocytes, fibroblasts, amnioticcells, osteoblasts, muscle cells, cardiac cells, etc.) and the like.Cells in an isolated placental stem cell population can be combined witha plurality of cells of another type in ratios of about 100,000,000:1,50,000,000:1, 20,000,000:1, 10,000,000:1, 5,000,000:1, 2,000,000:1,1,000,000:1, 500,000:1, 200,000:1, 100,000:1, 50,000:1, 20,000:1,10,000:1, 5,000:1, 2,000:1, 1,000:1, 500:1, 200:1, 100:1, 50:1, 20:1,10:1, 5:1, 2:1, 1:1; 1:2; 1:5; 1:10; 1:100; 1:200; 1:500; 1:1,000;1:2,000; 1:5,000; 1:10,000; 1:20,000; 1:50,000; 1:100,000; 1:500,000;1:1,000,000; 1:2,000,000; 1:5,000,000; 1:10,000,000; 1:20,000,000;1:50,000,000; or about 1:100,000,000, comparing numbers of totalnucleated cells in each population. Cells in an isolated placental stemcell population can be combined with a plurality of cells of a pluralityof cell types, as well.

In one, an isolated population of placental stem cells is combined witha plurality of hematopoietic stem cells. Such hematopoietic stem cellscan be, for example, contained within unprocessed placental, umbilicalcord blood or peripheral blood; in total nucleated cells from placentalblood, umbilical cord blood or peripheral blood; in an isolatedpopulation of CD34⁺ cells from placental blood, umbilical cord blood orperipheral blood; in unprocessed bone marrow; in total nucleated cellsfrom bone marrow; in an isolated population of CD34⁺ cells from bonemarrow, or the like.

5.5 Preservation of Placental Stem Cells

Placental stem cells can be preserved, that is, placed under conditionsthat allow for long-term storage, or conditions that inhibit cell deathby, e.g., apoptosis or necrosis.

Placental stem cells can be preserved using, e.g., a compositioncomprising an apoptosis inhibitor, necrosis inhibitor and/or anoxygen-carrying perfluorocarbon, as described in related U.S.Provisional Application No. 60/754,969, entitled “Improved Compositionfor Collecting and Preserving Placental Stem Cells and Methods of Usingthe Composition” filed on Dec. 25, 2005. In one embodiment, theinvention provides a method of preserving a population of stem cellscomprising contacting said population of stem cells with a stem cellcollection composition comprising an inhibitor of apoptosis and anoxygen-carrying perfluorocarbon, wherein said inhibitor of apoptosis ispresent in an amount and for a time sufficient to reduce or preventapoptosis in the population of stem cells, as compared to a populationof stem cells not contacted with the inhibitor of apoptosis. In aspecific embodiment, said inhibitor of apoptosis is a caspase inhibitor.In another specific embodiment, said inhibitor of apoptosis is a JNKinhibitor. In a more specific embodiment, said JNK inhibitor does notmodulate differentiation or proliferation of said stem cells. In anotherembodiment, said stem cell collection composition comprises saidinhibitor of apoptosis and said oxygen-carrying perfluorocarbon inseparate phases. In another embodiment, said stem cell collectioncomposition comprises said inhibitor of apoptosis and saidoxygen-carrying perfluorocarbon in an emulsion. In another embodiment,the stem cell collection composition additionally comprises anemulsifier, e.g., lecithin. In another embodiment, said apoptosisinhibitor and said perfluorocarbon are between about 0° C. and about 25°C. at the time of contacting the stem cells. In another more specificembodiment, said apoptosis inhibitor and said perfluorocarbon arebetween about 2° C. and 10° C., or between about 2° C. and about 5° C.,at the time of contacting the stem cells. In another more specificembodiment, said contacting is performed during transport of saidpopulation of stem cells. In another more specific embodiment, saidcontacting is performed during freezing and thawing of said populationof stem cells.

In another embodiment, the invention provides a method of preserving apopulation of placental stem cells comprising contacting said populationof stem cells with an inhibitor of apoptosis and an organ-preservingcompound, wherein said inhibitor of apoptosis is present in an amountand for a time sufficient to reduce or prevent apoptosis in thepopulation of stem cells, as compared to a population of stem cells notcontacted with the inhibitor of apoptosis. In a specific embodiment, theorgan-preserving compound is UW solution (described in U.S. Pat. No.4,798,824; also known as ViaSpan; see also Southard et al.,Transplantation 49(2):251-257 (1990)) or a solution described in Sternet al., U.S. Pat. No. 5,552,267. In another embodiment, saidorgan-preserving compound is hydroxyethyl starch, lactobionic acid,raffinose, or a combination thereof. In another embodiment, the stemcell collection composition additionally comprises an oxygen-carryingperfluorocarbon, either in two phases or as an emulsion.

In another embodiment of the method, placental stem cells are contactedwith a stem cell collection composition comprising an apoptosisinhibitor and oxygen-carrying perfluorocarbon, organ-preservingcompound, or combination thereof, during perfusion. In anotherembodiment, said stem cells are contacted during a process of tissuedisruption, e.g., enzymatic digestion. In another embodiment, placentalstem cells are contacted with said stem cell collection compound aftercollection by perfusion, or after collection by tissue disruption, e.g.,enzymatic digestion.

Typically, during placental cell collection, enrichment and isolation,it is preferable to minimize or eliminate cell stress due to hypoxia andmechanical stress. In another embodiment of the method, therefore, astem cell, or population of stem cells, is exposed to a hypoxiccondition during collection, enrichment or isolation for less than sixhours during said preservation, wherein a hypoxic condition is aconcentration of oxygen that is less than normal blood oxygenconcentration. In a more specific embodiment, said population of stemcells is exposed to said hypoxic condition for less than two hoursduring said preservation. In another more specific embodiment, saidpopulation of stem cells is exposed to said hypoxic condition for lessthan one hour, or less than thirty minutes, or is not exposed to ahypoxic condition, during collection, enrichment or isolation. Inanother specific embodiment, said population of stem cells is notexposed to shear stress during collection, enrichment or isolation.

The placental stem cells of the invention can be cryopreserved, e.g., incryopreservation medium in small containers, e.g., ampoules. Suitablecryopreservation medium includes, but is not limited to, culture mediumincluding, e.g., growth medium, or cell freezing medium, for examplecommercially available cell freezing medium, e.g., C2695, C2639 or C6039(Sigma). Cryopreservation medium preferably comprises DMSO(dimethylsulfoxide), at a concentration of, e.g., about 10% (v/v).Cryopreservation medium may comprise additional agents, for example,methylcellulose and/or glycerol. Placental stem cells are preferablycooled at about 1° C./min during cryopreservation. A preferredcryopreservation temperature is about −80° C. to about −180° C.,preferably about −125° C. to about −140° C. Cryopreserved cells can betransferred to liquid nitrogen prior to thawing for use. In someembodiments, for example, once the ampoules have reached about −90° C.,they are transferred to a liquid nitrogen storage area. Cryopreservedcells preferably are thawed at a temperature of about 25° C. to about40° C., preferably to a temperature of about 37° C.

5.6 Uses of Placental Stem Cells

5.6.1 Compositions Comprising Placental Stem Cells

The methods of immunosuppression of the present invention can usecompositions comprising placental stem cells, or biomolecules therefrom.In the same manner, the pluralities and populations of placental stemcells of the present invention can be combined with anyphysiologically-acceptable or medically-acceptable compound, compositionor device for use in, e.g., research or therapeutics.

5.6.1.1 Cryopreserved Placental Stem Cells

The immunosuppressive placental stem cell populations of the inventioncan be preserved, for example, cryopreserved for later use. Methods forcryopreservation of cells, such as stem cells, are well known in theart. Placental stem cell populations can be prepared in a form that iseasily administrable to an individual. For example, the inventionprovides a placental stem cell population that is contained within acontainer that is suitable for medical use. Such a container can be, forexample, a sterile plastic bag, flask, jar, or other container fromwhich the placental stem cell population can be easily dispensed. Forexample, the container can be a blood bag or other plastic,medically-acceptable bag suitable for the intravenous administration ofa liquid to a recipient. The container is preferably one that allows forcryopreservation of the combined stem cell population.

Cryopreserved immunosuppressive placental stem cell populations cancomprise placental stem cells derived from a single donor, or frommultiple donors. The placental stem cell population can be completelyHLA-matched to an intended recipient, or partially or completelyHLA-mismatched.

Thus, in one embodiment, the invention provides a composition comprisingan immunosuppressive placental stem cell population in a container. In aspecific embodiment, the stem cell population is cryopreserved. Inanother specific embodiment, the container is a bag, flask, or jar. Inmore specific embodiment, said bag is a sterile plastic bag. In a morespecific embodiment, said bag is suitable for, allows or facilitatesintravenous administration of said placental stem cell population. Thebag can comprise multiple lumens or compartments that are interconnectedto allow mixing of the placental stem cells and one or more othersolutions, e.g., a drug, prior to, or during, administration. In anotherspecific embodiment, the composition comprises one or more compoundsthat facilitate cryopreservation of the combined stem cell population.In another specific embodiment, said placental stem cell population iscontained within a physiologically-acceptable aqueous solution. In amore specific embodiment, said physiologically-acceptable aqueoussolution is a 0.9% NaCl solution. In another specific embodiment, saidplacental stem cell population comprises placental cells that areHLA-matched to a recipient of said stem cell population. In anotherspecific embodiment, said combined stem cell population comprisesplacental cells that are at least partially HLA-mismatched to arecipient of said stem cell population. In another specific embodiment,said placental stem cells are derived from a plurality of donors.

5.6.1.2 Pharmaceutical Compositions

Immunosuppressive populations of placental stem cells, or populations ofcells comprising placental stem cells, can be formulated intopharmaceutical compositions for use in vivo. Such pharmaceuticalcompositions comprise a population of placental stem cells, or apopulation of cells comprising placental stem cells, in apharmaceutically-acceptable carrier, e.g., a saline solution or otheraccepted physiologically-acceptable solution for in vivo administration.Pharmaceutical compositions of the invention can comprise any of theplacental stem cell populations, or placental stem cell types, describedelsewhere herein. The pharmaceutical compositions can comprise fetal,maternal, or both fetal and maternal placental stem cells. Thepharmaceutical compositions of the invention can further compriseplacental stem cells obtained from a single individual or placenta, orfrom a plurality of individuals or placentae.

The pharmaceutical compositions of the invention can comprise anyimmunosuppressive number of placental stem cells. For example, a singleunit dose of placental stem cells can comprise, in various embodiments,about, at least, or no more than 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, 1×10⁷,5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹ or moreplacental stem cells.

The pharmaceutical compositions of the invention comprise populations ofcells that comprise 50% viable cells or more (that is, at least 50% ofthe cells in the population are functional or living). Preferably, atleast 60% of the cells in the population are viable. More preferably, atleast 70%, 80%, 90%, 95%, or 99% of the cells in the population in thepharmaceutical composition are viable.

The pharmaceutical compositions of the invention can comprise one ormore compounds that, e.g., facilitate engraftment (e.g., anti-T-cellreceptor antibodies, an immunosuppressant, or the like); stabilizerssuch as albumin, dextran 40, gelatin, hydroxyethyl starch, and the like.

5.6.1.3 Placental Stem Cell Conditioned Media

The placental stem cells of the invention can be used to produceconditioned medium that is immunosuppressive, that is, medium comprisingone or more biomolecules secreted or excreted by the stem cells thathave a detectable immunosuppressive effect on a plurality of one or moretypes of immune cells. In various embodiments, the conditioned mediumcomprises medium in which placental stem cells have grown for at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days. In otherembodiments, the conditioned medium comprises medium in which placentalstem cells have grown to at least 30%, 40%, 50%, 60%, 70%, 80%, 90%confluence, or up to 100% confluence. Such conditioned medium can beused to support the culture of a separate population of placental stemcells, or stem cells of another kind. In another embodiment, theconditioned medium comprises medium in which placental stem cells havebeen differentiated into an adult cell type. In another embodiment, theconditioned medium of the invention comprises medium in which placentalstem cells and non-placental stem cells have been cultured.

Thus, in one embodiment, the invention provides a composition comprisingculture medium from a culture of placental stem cells, wherein saidplacental stem cells (a) adhere to a substrate; (b) express CD200 andHLA-G, or express CD73, CD105, and CD200, or express CD200 and OCT-4, orexpress CD73, CD105, and HLA-G, or express CD73 and CD105 and facilitatethe formation of one or more embryoid-like bodies in a population ofplacental cells that comprise the placental stem cells, when saidpopulation is cultured under conditions that allow formation ofembryoid-like bodies, or express OCT-4 and facilitate the formation ofone or more embryoid-like bodies in a population of placental cells thatcomprise the placental stem cells when said population is cultured underconditions that allow formation of embryoid-like bodies; and (c)detectably suppress CD4⁺ or CD8⁺ T cell proliferation in an MLR (mixedlymphocyte reaction), wherein said culture of placental stem cells hasbeen cultured in said medium for 24 hours or more. In a specificembodiment, the composition further comprises a plurality of saidplacental stem cells. In another specific embodiment, the compositioncomprises a plurality of non-placental cells. In a more specificembodiment, said non-placental cells comprise CD34⁺ cells, e.g.,hematopoietic progenitor cells, such as peripheral blood hematopoieticprogenitor cells, cord blood hematopoietic progenitor cells, orplacental blood hematopoietic progenitor cells. The non-placental cellscan also comprise other stem cells, such as mesenchymal stem cells,e.g., bone marrow-derived mesenchymal stem cells. The non-placentalcells can also be one or more types of adult cells or cell lines. Inanother specific embodiment, the composition comprises ananti-proliferative agent, e.g., an anti-MIP-1α or anti-MIP-1 antibody.

5.6.1.4 Matrices Comprising Placental Stem Cells

The invention further comprises matrices, hydrogels, scaffolds, and thelike that comprise an immunosuppresive population of placental stemcells.

Placental stem cells of the invention can be seeded onto a naturalmatrix, e.g., a placental biomaterial such as an amniotic membranematerial. Such an amniotic membrane material can be, e.g., amnioticmembrane dissected directly from a mammalian placenta; fixed orheat-treated amniotic membrane, substantially dry (i.e., <20% H₂O)amniotic membrane, chorionic membrane, substantially dry chorionicmembrane, substantially dry amniotic and chorionic membrane, and thelike. Preferred placental biomaterials on which placental stem cells canbe seeded are described in Hariri, U.S. Application Publication No.2004/0048796.

Placental stem cells of the invention can be suspended in a hydrogelsolution suitable for, e.g., injection. Suitable hydrogels for suchcompositions include self-assembling peptides, such as RAD16. In oneembodiment, a hydrogel solution comprising the cells can be allowed toharden, for instance in a mold, to form a matrix having cells dispersedtherein for implantation. Placental stem cells in such a matrix can alsobe cultured so that the cells are mitotically expanded prior toimplantation. The hydrogel is, e.g., an organic polymer (natural orsynthetic) that is cross-linked via covalent, ionic, or hydrogen bondsto create a three-dimensional open-lattice structure that entraps watermolecules to form a gel. Hydrogel-forming materials includepolysaccharides such as alginate and salts thereof, peptides,polyphosphazines, and polyacrylates, which are crosslinked ionically, orblock polymers such as polyethylene oxide-polypropylene glycol blockcopolymers which are crosslinked by temperature or pH, respectively. Insome embodiments, the hydrogel or matrix of the invention isbiodegradable.

In some embodiments of the invention, the formulation comprises an insitu polymerizable gel (see., e.g., U.S. Patent Application Publication2002/0022676; Anseth et al., J. Control Release, 78(1-3):199-209 (2002);Wang et al., Biomaterials, 24(22):3969-80 (2003).

In some embodiments, the polymers are at least partially soluble inaqueous solutions, such as water, buffered salt solutions, or aqueousalcohol solutions, that have charged side groups, or a monovalent ionicsalt thereof. Examples of polymers having acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, poly(vinyl acetate), and sulfonated polymers, such as sulfonatedpolystyrene. Copolymers having acidic side groups formed by reaction ofacrylic or methacrylic acid and vinyl ether monomers or polymers canalso be used. Examples of acidic groups are carboxylic acid groups,sulfonic acid groups, halogenated (preferably fluorinated) alcoholgroups, phenolic OH groups, and acidic OH groups.

The placental stem cells of the invention or co-cultures thereof can beseeded onto a three-dimensional framework or scaffold and implanted invivo. Such a framework can be implanted in combination with any one ormore growth factors, cells, drugs or other components that stimulatetissue formation or otherwise enhance or improve the practice of theinvention.

Examples of scaffolds that can be used in the present invention includenonwoven mats, porous foams, or self assembling peptides. Nonwoven matscan be formed using fibers comprised of a synthetic absorbable copolymerof glycolic and lactic acids (e.g., PGA/PLA) (VICRYL, Ethicon, Inc.,Somerville, N.J.). Foams, composed of, e.g.,poly(s-caprolactone)/poly(glycolic acid) (PCL/PGA) copolymer, formed byprocesses such as freeze-drying, or lyophilization (see, e.g., U.S. Pat.No. 6,355,699), can also be used as scaffolds.

Placental stem cells of the invention can also be seeded onto, orcontacted with, a physiologically-acceptable ceramic material including,but not limited to, mono-, di-, tri-, alpha-tri-, beta-tri-, andtetra-calcium phosphate, hydroxyapatite, fluoroapatites, calciumsulfates, calcium fluorides, calcium oxides, calcium carbonates,magnesium calcium phosphates, biologically active glasses such asBIOGLASS®, and mixtures thereof. Porous biocompatible ceramic materialscurrently commercially available include SURGIBONE® (CanMedica Corp.,Canada), ENDOBON® (Merck Biomaterial France, France), CEROS® (Mathys,AG, Bettlach, Switzerland), and mineralized collagen bone graftingproducts such as HEALOS™ (DePuy, Inc., Raynham, Mass.) and VITOSS®,RHAKOSS™, and CORTOSS® (Orthovita, Malvern, Pa.). The framework can be amixture, blend or composite of natural and/or synthetic materials.

In another embodiment, placental stem cells can be seeded onto, orcontacted with, a felt, which can be, e.g., composed of a multifilamentyarn made from a bioabsorbable material such as PGA, PLA, PCL copolymersor blends, or hyaluronic acid.

The placental stem cells of the invention can, in another embodiment, beseeded onto foam scaffolds that may be composite structures. Such foamscaffolds can be molded into a useful shape, such as that of a portionof a specific structure in the body to be repaired, replaced oraugmented. In some embodiments, the framework is treated, e.g., with0.1M acetic acid followed by incubation in polylysine, PBS, and/orcollagen, prior to inoculation of the cells of the invention in order toenhance cell attachment. External surfaces of a matrix may be modifiedto improve the attachment or growth of cells and differentiation oftissue, such as by plasma-coating the matrix, or addition of one or moreproteins (e.g., collagens, elastic fibers, reticular fibers),glycoproteins, glycosaminoglycans (e.g., heparin sulfate,chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratinsulfate, etc.), a cellular matrix, and/or other materials such as, butnot limited to, gelatin, alginates, agar, agarose, and plant gums, andthe like.

In some embodiments, the scaffold comprises, or is treated with,materials that render it non-thrombogenic. These treatments andmaterials may also promote and sustain endothelial growth, migration,and extracellular matrix deposition. Examples of these materials andtreatments include but are not limited to natural materials such asbasement membrane proteins such as laminin and Type IV collagen,synthetic materials such as EPTFE, and segmented polyurethaneureasilicones, such as PURSPAN™ (The Polymer Technology Group, Inc.,Berkeley, Calif.). The scaffold can also comprise anti-thrombotic agentssuch as heparin; the scaffolds can also be treated to alter the surfacecharge (e.g., coating with plasma) prior to seeding with placental stemcells.

5.6.2 Immortalized Placental Stem Cell Lines

Mammalian placental cells can be conditionally immortalized bytransfection with any suitable vector containing a growth-promotinggene, that is, a gene encoding a protein that, under appropriateconditions, promotes growth of the transfected cell, such that theproduction and/or activity of the growth-promoting protein isregulatable by an external factor. In a preferred embodiment thegrowth-promoting gene is an oncogene such as, but not limited to, v-myc,N-myc, c-myc, p53, SV40 large T antigen, polyoma large T antigen, E1aadenovirus or E7 protein of human papillomavirus.

External regulation of the growth-promoting protein can be achieved byplacing the growth-promoting gene under the control of anexternally-regulatable promoter, e.g., a promoter the activity of whichcan be controlled by, for example, modifying the temperature of thetransfected cells or the composition of the medium in contact with thecells. In one embodiment, a tetracycline (tet)-controlled geneexpression system can be employed (see Gossen et al., Proc. Natl. Acad.Sci. USA 89:5547-5551, 1992; Hoshimaru et al., Proc. Natl. Acad. Sci.USA 93:1518-1523, 1996). In the absence of tet, a tet-controlledtransactivator (tTA) within this vector strongly activates transcriptionfrom ph_(CMV*-1), a minimal promoter from human cytomegalovirus fused totet operator sequences. tTA is a fusion protein of the repressor (tetR)of the transposon-10-derived tet resistance operon of Escherichia coliand the acidic domain of VP 16 of herpes simplex virus. Low, non-toxicconcentrations of tet (e.g., 0.01-1.0 μg/mL) almost completely abolishtransactivation by tTA.

In one embodiment, the vector further contains a gene encoding aselectable marker, e.g., a protein that confers drug resistance. Thebacterial neomycin resistance gene (neo^(R)) is one such marker that maybe employed within the present invention. Cells carrying neo^(R) may beselected by means known to those of ordinary skill in the art, such asthe addition of, e.g., 100-200 μg/mL G418 to the growth medium.

Transfection can be achieved by any of a variety of means known to thoseof ordinary skill in the art including, but not limited to, retroviralinfection. In general, a cell culture may be transfected by incubationwith a mixture of conditioned medium collected from the producer cellline for the vector and DMEM/F12 containing N2 supplements. For example,a placental cell culture prepared as described above may be infectedafter, e.g., five days in vitro by incubation for about 20 hours in onevolume of conditioned medium and two volumes of DMEM/F12 containing N2supplements. Transfected cells carrying a selectable marker may then beselected as described above.

Following transfection, cultures are passaged onto a surface thatpermits proliferation, e.g., allows at least 30% of the cells to doublein a 24 hour period. Preferably, the substrate is apolyornithine/laminin substrate, consisting of tissue culture plasticcoated with polyornithine (10 μg/mL) and/or laminin (10 μg/mL), apolylysine/laminin substrate or a surface treated with fibronectin.Cultures are then fed every 3-4 days with growth medium, which may ormay not be supplemented with one or more proliferation-enhancingfactors. Proliferation-enhancing factors may be added to the growthmedium when cultures are less than 50% confluent.

The conditionally-immortalized placental stem cell lines can be passagedusing standard techniques, such as by trypsinization, when 80-95%confluent. Up to approximately the twentieth passage, it is, in someembodiments, beneficial to maintain selection (by, for example, theaddition of G418 for cells containing a neomycin resistance gene). Cellsmay also be frozen in liquid nitrogen for long-term storage.

Clonal cell lines can be isolated from a conditionally-immortalizedhuman placental stem cell line prepared as described above. In general,such clonal cell lines may be isolated using standard techniques, suchas by limit dilution or using cloning rings, and expanded. Clonal celllines may generally be fed and passaged as described above.

Conditionally-immortalized human placental stem cell lines, which may,but need not, be clonal, may generally be induced to differentiate bysuppressing the production and/or activity of the growth-promotingprotein under culture conditions that facilitate differentiation. Forexample, if the gene encoding the growth-promoting protein is under thecontrol of an externally-regulatable promoter, the conditions, e.g.,temperature or composition of medium, may be modified to suppresstranscription of the growth-promoting gene. For thetetracycline-controlled gene expression system discussed above,differentiation can be achieved by the addition of tetracycline tosuppress transcription of the growth-promoting gene. In general, 1 μg/mLtetracycline for 4-5 days is sufficient to initiate differentiation. Topromote further differentiation, additional agents may be included inthe growth medium.

5.6.3 Assays

The placental stem cells for the present invention can be used in assaysto determine the influence of culture conditions, environmental factors,molecules (e.g., biomolecules, small inorganic molecules. etc.) and thelike on stem cell proliferation, expansion, and/or differentiation,compared to placental stem cells not exposed to such conditions.

In a preferred embodiment, the placental stem cells of the presentinvention are assayed for changes in proliferation, expansion ordifferentiation upon contact with a molecule. In one embodiment, forexample, the invention provides a method of identifying a compound thatmodulates the proliferation of a plurality of placental stem cells,comprising contacting said plurality of stem cells with said compoundunder conditions that allow proliferation, wherein if said compoundcauses a detectable change in proliferation of said plurality of stemcells compared to a plurality of stem cells not contacted with saidcompound, said compound is identified as a compound that modulatesproliferation of placental stem cells. In a specific embodiment, saidcompound is identified as an inhibitor of proliferation. In anotherspecific embodiment, said compound is identified as an enhancer ofproliferation.

In another embodiment, the invention provides a method of identifying acompound that modulates the expansion of a plurality of placental stemcells, comprising contacting said plurality of stem cells with saidcompound under conditions that allow expansion, wherein if said compoundcauses a detectable change in expansion of said plurality of stem cellscompared to a plurality of stem cells not contacted with said compound,said compound is identified as a compound that modulates expansion ofplacental stem cells. In a specific embodiment, said compound isidentified as an inhibitor of expansion. In another specific embodiment,said compound is identified as an enhancer of expansion.

In another embodiment, the invention provides a method of identifying acompound that modulates the differentiation of a placental stem cell,comprising contacting said stem cells with said compound underconditions that allow differentiation, wherein if said compound causes adetectable change in differentiation of said stem cells compared to astem cell not contacted with said compound, said compound is identifiedas a compound that modulates proliferation of placental stem cells. In aspecific embodiment, said compound is identified as an inhibitor ofdifferentiation. In another specific embodiment, said compound isidentified as an enhancer of differentiation.

5.6.4 Placental Stem Cell Bank

Stem cells from postpartum placentas can be cultured in a number ofdifferent ways to produce a set of lots, e.g., a set ofindividually-administrable doses, of placental stem cells. Such lotscan, for example, be obtained from stem cells from placental perfusateor from enzyme-digested placental tissue. Sets of lots of placental stemcells, obtained from a plurality of placentas, can be arranged in a bankof placental stem cells for, e.g., long-term storage. Generally,adherent stem cells are obtained from an initial culture of placentalmaterial to form a seed culture, which is expanded under controlledconditions to form populations of cells from approximately equivalentnumbers of doublings. Lots are preferably derived from the tissue of asingle placenta, but can be derived from the tissue of a plurality ofplacentas.

In one embodiment, stem cell lots are obtained as follows. Placentaltissue is first disrupted, e.g., by mincing, digested with a suitableenzyme, e.g., collagenase (see Section 5.2.3, above). The placentaltissue preferably comprises, e.g., the entire amnion, entire chorion, orboth, from a single placenta, but can comprise only a part of either theamnion or chorion. The digested tissue is cultured, e.g., for about 1-3weeks, preferably about 2 weeks. After removal of non-adherent cells,high-density colonies that form are collected, e.g., by trypsinization.These cells are collected and resuspended in a convenient volume ofculture medium, and defined as Passage 0 cells.

Passage 0 cells are then used to seed expansion cultures. Expansioncultures can be any arrangement of separate cell culture apparatuses,e.g., a Cell Factory by NUNC™. Cells in the Passage 0 culture can besubdivided to any degree so as to seed expansion cultures with, e.g.,1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴,1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴, 9×10⁴, or 10×10⁴stem cells. Preferably, from about 2×10⁴ to about 3×10⁴ Passage 0 cellsare used to seed each expansion culture. The number of expansioncultures can depend upon the number of Passage 0 cells, and may begreater or fewer in number depending upon the particular placenta(s)from which the stem cells are obtained.

Expansion cultures are grown until the density of cells in culturereaches a certain value, e.g., about 1×10⁵ cells/cm². Cells can eitherbe collected and cryopreserved at this point, or passaged into newexpansion cultures as described above. Cells can be passaged, e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 timesprior to use. A record of the cumulative number of population doublingsis preferably maintained during expansion culture(s). The cells from aPassage 0 culture can be expanded for 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 doublings, orup to 60 doublings. Preferably, however, the number of populationdoublings, prior to dividing the population of cells into individualdoses, is between about 15 and about 30, preferably about 20 doublings.The cells can be culture continuously throughout the expansion process,or can be frozen at one or more points during expansion.

Cells to be used for individual doses can be frozen, e.g., cryopreservedfor later use. Individual doses can comprise, e.g., about 1 million toabout 100 million cells per ml, and can comprise between about 10⁶ andabout 10⁹ cells in total.

In a specific embodiment, of the method, Passage 0 cells are culturedfor approximately 4 doublings, then frozen in a first cell bank. Cellsfrom the first cell bank are frozen and used to seed a second cell bank,the cells of which are expanded for about another eight doublings. Cellsat this stage are collected and frozen and used to seed new expansioncultures that are allowed to proceed for about eight additionaldoublings, bringing the cumulative number of cell doublings to about 20.Cells at the intermediate points in passaging can be frozen in units ofabout 100,000 to about 10 million cells per ml, preferably about 1million cells per ml for use in subsequent expansion culture. Cells atabout 20 doublings can be frozen in individual doses of between about 1million to about 100 million cells per ml for administration or use inmaking a stem cell-containing composition.

In a preferred embodiment, the donor from which the placenta is obtained(e.g., the mother) is tested for at least one pathogen. If the mothertests positive for a tested pathogen, the entire lot from the placentais discarded. Such testing can be performed at any time duringproduction of placental stem cell lots, including before or afterestablishment of Passage 0 cells, or during expansion culture. Pathogensfor which the presence is tested can include, without limitation,hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, humanimmunodeficiency virus (types I and II), cytomegalovirus, herpesvirus,and the like.

5.6.5 Treatment of Multiple Sclerosis

In another aspect, the invention provides a method of treating anindividual having multiple sclerosis, or a symptom associated withmultiple sclerosis, comprising administering to the individual aplurality of placental stem cells in an amount and for a time sufficientto detectably modulate, e.g., suppress an immune response in theindividual.

Multiple sclerosis (MS) is a chronic, recurrent inflammatory disease ofthe central nervous system. The disease results in injury to the myelinsheaths surrounding CNS and PNS axons, oligodendrocytes, and the nervecells themselves. The disease is mediated by autoreactive T cells,particularly CD4⁺ T cells, that proliferate, cross the blood-brainbarrier, and enter the CNS under the influence of cellular adhesionmolecules and pro-inflammatory cytokines. The symptoms of MS includesensory disturbances in the limbs, optic nerve dysfunction, pyramidaltract dysfunction, bladder dysfunction, bowel dysfunction, sexualdysfunction, ataxia, and diplopia.

Four different types or clinical courses of MS have been identified. Thefirst, relapsing/remitting MS (RRMS) is characterized by self-limitingattacks of neurological dysfunction that manifest acutely, over thecourse of days to weeks, followed by a period of recovery, sometimesincomplete, over several months. The second type, secondary progressiveMS (SPMS), begins as RRMS but changes such that the clinical coursebecomes characterized by a steady deterioration in function unrelated toacute attacks. The third, primary progressive MS (PPMS), ischaracterized by a steady decline in function from onset, with no acuteattacks. The fourth type, progressive/relapsing MS (PRMS), also beginswith a progressive course, with occasional attacks superimposed on theprogressive decline in function.

Persons having MS are generally evaluated using a motor skillsassessment, optionally with an MRI. For example, one motor skillsassessment, the expanded disability status scale, scores gradations inan affected individual's abilities, as follows:

-   -   0.0 Normal neurological examination    -   1.0 No disability, minimal signs in one FS    -   1.5 No disability, minimal signs in more than one FS    -   2.0 Minimal disability in one FS    -   2.5 Mild disability in one FS or minimal disability in two FS    -   3.0 Moderate disability in one FS, or mild disability in three        or four FS. Fully ambulatory.    -   3.5 Fully ambulatory but with moderate disability in one FS and        more than minimal disability in several others    -   4.0 Fully ambulatory without aid, self-sufficient, up and about        some 12 hours a day despite relatively severe disability; able        to walk without aid or rest some 500 meters    -   4.5 Fully ambulatory without aid, up and about much of the day,        able to work a full day, may otherwise have some limitation of        full activity or require minimal assistance; characterized by        relatively severe disability; able to walk without aid or rest        some 300 meters.    -   5.0 Ambulatory without aid or rest for about 200 meters;        disability severe enough to impair full daily activities (work a        full day without special provisions)    -   5.5 Ambulatory without aid or rest for about 100 meters;        disability severe enough to preclude full daily activities    -   6.0 Intermittent or unilateral constant assistance (cane,        crutch, brace) required to walk about 100 meters with or without        resting    -   6.5 Constant bilateral assistance (canes, crutches, braces)        required to walk about 20 meters without resting    -   7.0 Unable to walk beyond approximately five meters even with        aid, essentially restricted to wheelchair; wheels self in        standard wheelchair and transfers alone; up and about in        wheelchair some 12 hours a day    -   7.5 Unable to take more than a few steps; restricted to        wheelchair; may need aid in transfer; wheels self but cannot        carry on in standard wheelchair a full day; May require        motorized wheelchair    -   8.0 Essentially restricted to bed or chair or perambulated in        wheelchair, but may be out of bed itself much of the day;        retains many self-care functions; generally has effective use of        arms    -   8.5 Essentially restricted to bed much of day; has some        effective use of arms retains some self care functions    -   9.0 Confined to bed; can still communicate and eat.    -   9.5 Totally helpless bed patient; unable to communicate        effectively or eat/swallow    -   10.0 Death due to MS

In the above scoring system, “FS” refers to the eight functional systemsmeasured, including pyramidal, cerebellar, brainstem, sensory, bowel andbladder, visual, cerebral, and other systems.

Other, similar scoring systems are known, including the Scrippsneurological rating scale, the ambulatory index, and the multiplesclerosis functional composite score (MSFC).

The progress of MS has also been assessed by a determination of theattack rate.

The progress of MS has also been assessed by magnetic resonance imaging,which can detect neural lesions associated with MS (e.g., new lesions,enhancing lesions, or combined unique active lesions).

Thus, in one embodiment, the invention provides a method of treating anindividual having MS, e.g., and individual who has been diagnosed withMS, comprising administering to the individual a plurality of placentalstem cells sufficient to suppress an immune response in the individual.In a specific embodiment, the administering detectably improves one ormore symptoms of MS in the individual. In more specific embodiments, thesymptom is, e.g., one or more of a sensory disturbance in the limbs, anoptic nerve dysfunction, a pyramidal tract dysfunction, a bladderdysfunction, a bowel dysfunction, a sexual dysfunction, ataxia, ordiplopia. In another specific embodiment, said administering results inan improvement on the EDSS scale of at least one half point. In anotherspecific embodiment, said administering results in an improvement on theEDSS scale of at least one point. In another specific embodiment, saidadministering results in an improvement on the EDSS scale of at leasttwo points. In other specific embodiments, said administering results ina detectable improvement on a multiple sclerosis assessment scale or onan MRI.

MS has been treated with other therapeutic agents, for exampleimmunomodulatory or immunosuppressive agents, e.g., interferon beta(IFNβ), including IFNβ-1a and IFNβ-1b; gliatriamer acetate (COPAXONE®);cyclophosphamide; methotrexate; azathioprine (IMURAN®); cladribine(LEUSTATIN®); cyclosporine; mitoxantrone; and the like. MS has also beentreated with anti-inflammatory therapeutic agents, such asglucocorticoids, including adrenocorticotropic hormone (ACTH),methylprednisolone, dexamethasone, and the like. MS has also beentreated with other types of therapeutic agents, such as intravenousimmunoglobulin, plasma exchange, or sulfasalazine.

Thus, the invention further provides for the treatment of an individualhaving MS, e.g., an individual who has been diagnosed as having MS,comprising administering to the individual a plurality of placental stemcells sufficient to suppress an immune response in the individual,wherein the administering detectably improves one or more symptoms of MSin the individual, and one or more therapeutic agents. In oneembodiment, the therapeutic agent is a glucocorticoid. In specificembodiments, the glucocorticoid is adrenocorticotropic hormone (ACTH),methylprednisolone, or dexamethasone. In another embodiment, thetherapeutic agent is an immunomodulatory or immunosuppressive agent. Invarious specific embodiments, the immunomodulatory or immunosuppressiveagent is IFNβ-1a, IFN-1b, gliatriamer acetate, cyclophosphamide,methotrexate, azathioprine, cladribine, cyclosporine or mitoxantrone. Inother embodiments, the therapeutic agent is intravenous immunoglobulin,plasma exchange, or sulfasalazine. In another embodiment, the individualis administered any combination of the foregoing therapeutic agents.

An individual having MS, e.g., an individual diagnosed with MS, can betreated with a plurality of placental stem cells, and, optionally, oneor more therapeutic agents, at any time during the progression of thedisease. For example, the individual can be treated immediately afterdiagnosis, or within 1, 2, 3, 4, 5, 6 days of diagnosis, or within 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more weeks,or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years after diagnosis. Theindividual can be treated once, or multiple times during the clinicalcourse of the disease. The individual can be treated, as appropriate,during an acute attack, during remission, or during a chronicdegenerative phase. In another embodiment, the placental stem cells areadministered to a female having MS, post-partum, to maintain the stateof remission or reduced occurrence of relapse experienced duringpregnancy.

In one embodiment, the individual is administered a dose of about 300million placental stem cells. Dosage, however, can vary according to theindividual's physical characteristics, e.g., weight, and can range from1 million to 10 billion placental stem cells per does, preferablybetween 10 million and 1 billion per dose, or between 100 million and 50million placental stem cells per dose. The administration is preferablyintravenous, but can be by any art-accepted route for the administrationof live cells. In one embodiment, the placental stem cells are from acell bank.

6. EXAMPLES 6.1 Example 1 Culture of Placental Stem Cells

Placental stem cells are obtained from a post-partum mammalian placentaeither by perfusion or by physical disruption, e.g., enzymaticdigestion. The cells are cultured in a culture medium comprising 60%DMEM-LG (Gibco), 40% MCDB-201(Sigma), 2% fetal calf serum (FCS) (HycloneLaboratories), 1× insulin-transferrin-selenium (ITS), 1×lenolenic-acid-bovine-serum-albumin (LA-BSA), 10⁻⁹ M dexamethasone(Sigma), 10⁻⁴M ascorbic acid 2-phosphate (Sigma), epidermal growthfactor (EGF) 10 ng/ml (R&D Systems), platelet derived-growth factor(PDGF-BB) 10 ng/ml (R&D Systems), and 100 U penicillin/1000 Ustreptomycin.

The culture flask in which the cells are cultured is prepared asfollows. T75 flasks are coated with fibronectin (FN), by adding 5 ml PBScontaining 5 ng/ml human FN (Sigma F0895) to the flask. The flasks withFN solution are left at 37° C. for 30 min. The FN solution is thenremoved prior to cell culture. There is no need to dry the flasksfollowing treatment. Alternatively, the flasks are left in contact withthe FN solution at 4° C. overnight or longer; prior to culture, theflasks are warmed and the FN solution is removed.

Placental Stem Cells Isolated by Perfusion

Cultures of placental stem cells from placental perfusate areestablished as follows. Cells from a Ficoll gradient are seeded inFN-coated T75 flasks, prepared as above, at 50-100×10⁶ cells/flask in 15ml culture medium. Typically, 5 to 10 flasks are seeded. The flasks areincubated at 37° C. for 12-18 hrs to allow the attachment of adherentcells. 10 ml of warm PBS is added to each flask to remove cells insuspension, and mixed gently. 15 mL of the medium is then removed andreplaced with 15 ml fresh culture medium. All medium is changed 3-4 daysafter the start of culture. Subsequent culture medium changes areperformed, during which 50% or 7.5 ml of the medium is removed.

Starting at about day 12, the culture is checked under a microscope toexamine the growth of the adherent cell colonies. When cell culturesbecome approximately 80% confluent, typically between day 13 to day 18after the start of culture, adherent cells are harvested by trypsindigestion. Cells harvested from these primary cultures are designatedpassage 0 (zero).

Placental Stem Cells Isolated by Physical Disruption and EnzymaticDigestion

Placental stem cell cultures are established from digested placentaltissue as follows. The perfused placenta is placed on a sterile papersheet with the maternal side up. Approximately 0.5 cm of the surfacelayer on maternal side of placenta is scraped off with a blade, and theblade is used to remove a placental tissue block measuring approximately1×2×1 cm. This placenta tissue is then minced into approximately 1 mm³pieces. These pieces are collected into a 50 ml Falcon tube and digestedwith collagenase IA (2 mg/ml, Sigma) for 30 minutes, followed bytrypsin-EDTA (0.25%, GIBCO BRL) for 10 minutes, at 37° C. in water bath.The resulting solution is centrifuged at 400 g for 10 minutes at roomtemperature, and the digestion solution is removed. The pellet isresuspended to approximately 10 volumes with PBS (for example, a 5 mlpellet is resuspended with 45 ml PBS), and the tubes are centrifuged at400 g for 10 minutes at room temperature. The tissue/cell pellet isresuspended in 130 mL culture medium, and the cells are seeded at 13 mlper fibronectin-coated T-75 flask. Cells are incubated at 37° C. with ahumidified atmosphere with 5% CO₂. Placental Stem Cells are optionallycryopreserved at this stage.

Subculturing and Expansion of Placental Stem Cells

Cryopreserved cells are quickly thawed in a 37° C. water bath. Placentalstem cells are immediately removed from the cryovial with 10 ml warmmedium and transferred to a 15 ml sterile tube. The cells arecentrifuged at 400 g for 10 minutes at room temperature. The cells aregently resuspended in 10 ml of warm culture medium by pipetting, andviable cell counts are determined by Trypan blue exclusion. Cells arethen seeded at about 6000-7000 cells per cm² onto FN-coated flasks,prepared as above (approximately 5×10⁵ cells per T-75 flask). The cellsare incubated at 37° C., 5% CO₂ and 90% humidity. When the cells reached75-85% confluency, all of the spent media is aseptically removed fromthe flasks and discarded. 3 ml of 0.25% trypsin/EDTA (w/v) solution isadded to cover the cell layer, and the cells are incubated at 37° C., 5%CO₂ and 90% humidity for 5 minutes. The flask is tapped once or twice toexpedite cell detachment. Once >95% of the cells are rounded anddetached, 7 ml of warm culture medium is added to each T-75 flask, andthe solution is dispersed by pipetting over the cell layer surfaceseveral times.

After counting the cells and determining viability as above, the cellsare centrifuged at 1000 RPM for 5 minutes at room temperature. Cells arepassaged by gently resuspending the cell pellet from one T-75 flask withculture medium, and evenly plating the cells onto two FN-coated T-75flasks.

Using the above methods, populations of adherent placental stem cellsare identified that express markers CD105, CD117, CD33, CD73, CD29,CD44, CD10, CD90 and CD133. This population of cells did not expressCD34 or CD45. Some, but not all cultures of these placental stem cellsexpressed HLA-ABC and/or HLA-DR.

6.2 Example 2 Isolation of Placental Stem Cells from PlacentalStructures

6.2.1 Materials & Methods

6.2.1.1 Isolation of the Phenotype of Interest

Five distinct populations of placental cells were obtained from theplacentas of normal, full-term pregnancies. All donors provided fullwritten consent for the use of their placentas for research purposes.Five populations of cells were examined: placental cells from (1)placental perfusate (from perfusion of the placental vasculature); andenzymatic digestions of (2) amnion, (3) chorion, (4) amnion-chorionplate and (5) umbilical cord cells from enzymatic digestion. The varioustissues were cleaned in sterile PBS (Gibco-Invitrogen Corporation,Carlsbad, Calif.) and placed on separate sterile Petri dishes. Thevarious tissues were minced using a sterile surgical scalpel and placedinto 50 mL Falcon Conical tubes. The minced tissues were digested with1× Collagenase (Sigma-Aldrich, St. Louis, Mo.) for 20 minutes in a 37°C. water bath, centrifuged, and then digested with 0.25% Trypsin-EDTA(Gibco-Invitrogen Corp) for 10 minutes in a 37° C. water bath. Thevarious tissues were centrifuged after digestion and rinsed once withsterile PBS (Gibco-Invitrogen Corp). The reconstituted cells were thenfiltered twice, once with 100 μm cell strainers and once with 30 μMseparation filters, to remove any residual extracellular matrix orcellular debris.

6.2.1.2 Cellular Viability Assessment and Cell Counts

The manual trypan blue exclusion method was employed post digestion tocalculate cell counts and assess cellular viability. Cells were mixedwith Trypan Blue Dye (Sigma-Aldrich) at a ratio of 1:1, and the cellswere read on hemacytometer.

6.2.1.3 Cell Surface Marker Characterization

Cells that were HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ were selected forcharacterization. Cells having this phenotype were identified,quantified, and characterized by two of Becton-Dickinson flowcytometers, the FACSCalibur and the FACS Aria (Becton-Dickinson, SanJose, Calif., USA). The various placental cells were stained, at a ratioof about 10 μL of antibody per 1 million cells, for 30 minutes at roomtemperature on a shaker. The following anti-human antibodies were used:Fluorescein Isothiocyanate (FITC) conjugated monoclonal antibodiesagainst HLA-G (Serotec, Raleigh, N.C.), CD10 (BD ImmunocytometrySystems, San Jose, Calif.), CD44 (BD Biosciences Pharmingen, San Jose,Calif.), and CD105 (R&D Systems Inc., Minneapolis, Minn.); Phycoerythrin(PE) conjugated monoclonal antibodies against CD44, CD200, CD117, andCD13 (BD Biosciences Pharmingen); Phycoerythrin-Cy5 (PE Cy5) conjugatedStreptavidin and monoclonal antibodies against CD117 (BD BiosciencesPharmingen); Phycoerythrin-Cy7 (PE Cy7) conjugated monoclonal antibodiesagainst CD33 and CD10 (BD Biosciences); Allophycocyanin (APC) conjugatedstreptavidin and monoclonal antibodies against CD38 (BD BiosciencesPharmingen); and Biotinylated CD90 (BD Biosciences Pharmingen). Afterincubation, the cells were rinsed once to remove unbound antibodies andwere fixed overnight with 4% paraformaldehyde (USB, Cleveland, Ohio) at4° C. The following day, the cells were rinsed twice, filtered through a30 μm separation filter, and were run on the flow cytometer(s).

Samples that were stained with anti-mouse IgG antibodies (BD BiosciencesPharmingen) were used as negative controls and were used to adjust thePhoto Multiplier Tubes (PMTs). Samples that were single stained withanti-human antibodies were used as positive controls and were used toadjust spectral overlaps/compensations.

6.2.1.4 Cell Sorting and Culture

One set of placental cells (from perfusate, amnion, or chorion) wasstained with 7-Amino-Actinomycin D (7AAD; BD Biosciences Pharmingen) andmonoclonal antibodies specific for the phenotype of interest. The cellswere stained at a ratio of 10 μL of antibody per 1 million cells, andwere incubated for 30 minutes at room temperature on a shaker. Thesecells were then positively sorted for live cells expressing thephenotype of interest on the BD FACS Aria and plated into culture.Sorted (population of interest) and “All” (non-sorted) placental cellpopulations were plated for comparisons. The cells were plated onto afibronectin (Sigma-Aldrich) coated 96 well plate at the cell densitieslisted in Table 1 (cells/cm²). The cell density, and whether the celltype was plated in duplicate or triplicate, was determined and governedby the number of cells expressing the phenotype of interest.

TABLE I Cell plating densities 96 Well Plate Culture Density of PlatedCells Conditions Sorted All All Max. Density Cell Source A Set #1: 40.6K/cm²  40.6 K/cm²  93.8 K/cm² Set #2 40.6 K/cm²  40.6 K/cm²  93.8 K/cm²Set #3: 40.6 K/cm²  40.6 K/cm²  93.8 K/cm² Cell Source B Set #1: 6.3K/cm² 6.3 K/cm² 62.5 K/cm² Set #2 6.3 K/cm² 6.3 K/cm² 62.5 K/cm² CellSource C Set #1: 6.3 K/cm² 6.3 K/cm² 62.5 K/cm² Set #2 6.3 K/cm² 6.3K/cm² 62.5 K/cm²

Complete medium (60% DMEM-LG (Gibco) and 40% MCDB-201 (Sigma); 2% fetalcalf serum (Hyclone Labs.); 1× insulin-transferrin-selenium (ITS); 1×linoleic acid-bovine serum albumin (LA-BSA); 10⁻⁹ M dexamethasone(Sigma); 10⁻⁴ M ascorbic acid 2-phosphate (Sigma); epidermal growthfactor 10 ng/mL (R&D Systems); and platelet-derived growth factor(PDGF-BB) 10 ng/mL (R&D Systems)) was added to each well of the 96 wellplate and the plate was placed in a 5% CO₂/37° C. incubator. On day 7,100 μL of complete medium was added to each of the wells. The 96 wellplate was monitored for about two weeks and a final assessment of theculture was completed on day 12.

6.2.1.5 Data Analysis

FACSCalibur data was analyzed in FlowJo (Tree star, Inc) using standardgating techniques. The BD FACS Aria data was analyzed using the FACSDivasoftware (Becton-Dickinson). The FACS Aria data was analyzed usingdoublet discrimination gating to minimize doublets, as well as, standardgating techniques. All results were compiled in Microsoft Excel and allvalues, herein, are represented as average±standard deviation (number,standard error of mean).

6.2.2 Results

6.2.2.1 Cellular Viability

Post-digestion viability was assessed using the manual trypan blueexclusion method (FIG. 1). The average viability of cells obtained fromthe majority of the digested tissue (from amnion, chorion oramnion-chorion plate) was around 70%. Cells from amnion had an averageviability of 74.35%±10.31% (n=6, SEM=4.21), chorion had an averageviability of 78.18%±12.65% (n=4, SEM=6.32), amnion-chorion plate had anaverage viability of 69.05%±10.80% (n=4, SEM=5.40), and umbilical cordhad an average viability of 63.30%±20.13% (n=4, SEM=10.06). Cells fromperfusion, which did not undergo digestion, retained the highest averageviability, 89.98±6.39% (n=5, SEM=2.86).

6.2.2.2 Cell Quantification

The five distinct populations of placenta derived cells were analyzed todetermine the numbers of HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ cells. From theanalysis of the BD FACSCalibur data, it was observed that the amnion,perfusate, and chorion contained the greatest total number of thesecells, 30.72±21.80 cells (n=4, SEM=10.90), 26.92±22.56 cells (n=3,SEM=13.02), and 18.39±6.44 cells (n=2, SEM=4.55) respectively (data notshown). The amnion-chorion plate and umbilical cord contained the leasttotal number of cells expressing the phenotype of interest, 4.72±4.16cells (n=3, SEM=2.40) and 3.94±2.58 cells (n=3, SEM=1.49) respectively(data not shown).

Similarly, when the percent of total cells expressing the phenotype ofinterest was analyzed, it was observed that amnion and placentalperfusate contained the highest percentages of cells expressing thisphenotype (0.0319%±0.0202% (n=4, SEM=0.0101) and 0.0269%±0.0226% (n=3,SEM=0.0130) respectively (FIG. 2). Although umbilical cord contained asmall number of cells expressing the phenotype of interest (FIG. 2), itcontained the third highest percentage of cells expressing the phenotypeof interest, 0.020±0.0226% (n=3, SEM=0.0131) (FIG. 2). The chorion andamnion-chorion plate contained the lowest percentages of cellsexpressing the phenotype of interest, 0.0184±0.0064% (n=2, SEM=0.0046)and 0.0177±0.0173% (n=3, SEM=0.010) respectively (FIG. 2).

Consistent with the results of the BD FACSCalibur analysis, the BD FACSAria data also identified amnion, perfusate, and chorion as providinghigher numbers of HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ cells than the remainingsources. The average total number of cells expressing the phenotype ofinterest among amnion, perfusate, and chorion was 126.47±55.61 cells(n=15, SEM=14.36), 81.65±34.64 cells (n=20, SEM=7.75), and 51.47±32.41cells (n=15, SEM=8.37), respectively (data not shown). Theamnion-chorion plate and umbilical cord contained the least total numberof cells expressing the phenotype of interest, 44.89±37.43 cells (n=9,SEM=12.48) and 11.00±4.03 cells (n=9, SEM=1.34) respectively (data notshown).

BD FACS Aria data revealed that the B and A cell sources contained thehighest percentages of HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ cells, 0.1523±0.0227%(n=15, SEM=0.0059) and 0.0929±0.0419% (n=20, SEM=0.0094) respectively(FIG. 3). The D cell source contained the third highest percentage ofcells expressing the phenotype of interest, 0.0632±0.0333% (n=9,SEM=0.0111) (FIG. 3). The C and E cell sources contained the lowestpercentages of cells expressing the phenotype of interest,0.0623±0.0249% (n=15, SEM=0.0064) and 0.0457±0.0055% (n=9, SEM=0.0018)respectively (FIG. 3).

After HLA ABC⁻/CD45⁻/CD34⁻/CD133⁺ cells were identified and quantifiedfrom each cell source, its cells were further analyzed and characterizedfor their expression of cell surface markers HLA-G, CD10, CD13, CD33,CD38, CD44, CD90, CD105, CD117, CD200, and CD105.

6.2.2.3 Placental Perfusate-Derived Cells

Perfusate-derived cells were consistently positive for HLA-G, CD33,CD117, CD10, CD44, CD200, CD90, CD38, CD105, and CD13 (FIG. 4). Theaverage expression of each marker for perfusate-derived cells was thefollowing: 37.15%±38.55% (n=4, SEM=19.28) of the cells expressed HLA-G;36.37%±21.98% (n=7, SEM=8.31) of the cells expressed CD33; 39.39%±39.91%(n=4, SEM=19.96) of the cells expressed CD117; 54.97%±33.08% (n=4,SEM=16.54) of the cells expressed CD10; 36.79%±11.42% (n=4, SEM=5.71) ofthe cells expressed CD44; 41.83%±19.42% (n=3, SEM=11.21) of the cellsexpressed CD200; 74.25%±26.74% (n=3, SEM=15.44) of the cells expressedCD90; 35.10%±23.10% (n=3, SEM=13.34) of the cells expressed CD38;22.87%±6.87% (n=3, SEM=3.97) of the cells expressed CD105; and25.49%±9.84% (n=3, SEM=5.68) of the cells expressed CD13.

6.2.2.4 Amnion-Derived Cells

Amnion-derived cells were consistently positive for HLA-G, CD33, CD117,CD10, CD44, CD200, CD90, CD38, CD105, and CD13 (FIG. 5). The averageexpression of each marker for amnion-derived was the following:57.27%±41.11% (n=3, SEM=23.73) of the cells expressed HLA-G;16.23%±15.81% (n=6, SEM=6.46) of the cells expressed CD33; 62.32%±37.89%(n=3, SEM=21.87) of the cells expressed CD117; 9.71%±13.73% (n=3,SEM=7.92) of the cells expressed CD10; 27.03%±22.65% (n=3, SEM=13.08) ofthe cells expressed CD44; 6.42%±0.88% (n=2, SEM=0.62) of the cellsexpressed CD200; 57.61%±22.10% (n=2, SEM=15.63) of the cells expressedCD90; 63.76%±4.40% (n=2, SEM=3.11) of the cells expressed CD38;20.27%±5.88% (n=2, SEM=4.16) of the cells expressed CD105; and54.37%±13.29% (n=2, SEM=9.40) of the cells expressed CD13.

6.2.2.5 Chorion-Derived Cells

Chorion-derived cells were consistently positive for HLA-G, CD117, CD10,CD44, CD200, CD90, CD38, and CD13, while the expression of CD33, andCD105 varied (FIG. 6). The average expression of each marker for chorioncells was the following: 53.25%±32.87% (n=3, SEM=18.98) of the cellsexpressed HLA-G; 15.44%±11.17% (n=6, SEM=4.56) of the cells expressedCD33; 70.76%±11.87% (n=3, SEM=6.86) of the cells expressed CD117;35.84%±25.96% (n=3, SEM=14.99) of the cells expressed CD10; 28.76%±6.09%(n=3, SEM=3.52) of the cells expressed CD44; 29.20% 9.47% (n=2,SEM=6.70) of the cells expressed CD200; 54.88%±0.17% (n=2, SEM=0.12) ofthe cells expressed CD90; 68.63%±44.37% (n=2, SEM=31.37) of the cellsexpressed CD38; 23.81%±33.67% (n=2, SEM=23.81) of the cells expressedCD105; and 53.16%±62.70% (n=2, SEM=44.34) of the cells expressed CD13.

6.2.2.6 Amnion-Chorion Plate Placental Cells

Cells from amnion-chorion plate were consistently positive for HLA-G,CD33, CD117, CD10, CD44, CD200, CD90, CD38, CD105, and CD13 (FIG. 7).The average expression of each marker for amnion-chorion plate-derivedcells was the following: 78.52%±13.13% (n=2, SEM=9.29) of the cellsexpressed HLA-G; 38.33%±15.74% (n=5, SEM=7.04) of the cells expressedCD33; 69.56%±26.41% (n=2, SEM=18.67) of the cells expressed CD117;42.44%±53.12% (n=2, SEM=37.56) of the cells expressed CD10;32.47%±31.78% (n=2, SEM=22.47) of the cells expressed CD44; 5.56% (n=1)of the cells expressed CD200; 83.33% (n=1) of the cells expressed CD90;83.52% (n=1) of the cells expressed CD38; 7.25% (n=1) of the cellsexpressed CD105; and 81.16% (n=1) of the cells expressed CD13.

6.2.2.7 Umbilical Cord-Derived Cells

Umbilical cord-derived cells were consistently positive for HLA-G, CD33,CD90, CD38, CD105, and CD13, while the expression of CD117, CD10, CD44,and CD200 varied (FIG. 8). The average expression of each marker forumbilical cord-derived cells was the following: 62.50%±53.03% (n=2,SEM=37.50) of the cells expressed HLA-G; 25.67%±11.28% (n=5, SEM=5.04)of the cells expressed CD33; 44.45%±62.85% (n=2, SEM=44.45) of the cellsexpressed CD117; 8.33%±11.79% (n=2, SEM=8.33) of the cells expressedCD10; 21.43%±30.30% (n=2, SEM=21.43) of the cells expressed CD44; 0.0%(n=1) of the cells expressed CD200; 81.25% (n=1) of the cells expressedCD90; 64.29% (n=1) of the cells expressed CD38; 6.25% (n=1) of the cellsexpressed CD105; and 50.0% (n=1) of the cells expressed CD13.

A summary of all marker expression averages is shown in FIG. 9.

6.2.2.8 BD FACS Aria Sort Report

The three distinct populations of placental cells that expressed thegreatest percentages of HLA ABC. CD45, CD34, and CD133 (cells derivedfrom perfusate, amnion and chorion) were stained with 7AAD and theantibodies for these markers. The three populations were positivelysorted for live cells expressing the phenotype of interest. The resultsof the BD FACS Aria sort are listed in table 2.

TABLE 2 BD FACS Aria Sort Report Events Sorted (Phenotype of Cell SourceEvents Processed Interest) % Of Total Perfusate 135540110 51215 0.037786Amnion 7385933 4019 0.054414 Chorion 108498122 4016 0.003701

The three distinct populations of positively sorted cells (“sorted”) andtheir corresponding non-sorted cells were plated and the results of theculture were assessed on day 12 (Table 3). Sorted perfusate-derivedcells, plated at a cell density of 40,600/cm², resulted in small, round,non-adherent cells. Two out of the three sets of non-sortedperfusate-derived cells, each plated at a cell density of 40,600/cm²,resulted in mostly small, round, non-adherent cells with severaladherent cells located around the periphery of well. Non-sortedperfusate-derived cells, plated at a cell density of 93,800/cm²,resulted in mostly small, round, non-adherent cells with severaladherent cells located around the well peripheries.

Sorted amnion-derived cells, plated at a cell density of 6,300/cm²,resulted in small, round, non-adherent cells. Non-sorted amnion-derivedcells, plated at a cell density of 6,300/cm², resulted in small, round,non-adherent cells. Non-sorted amnion-derived cells plated at a celldensity of 62,500/cm² resulted in small, round, non-adherent cells.

Sorted chorion-derived cells, plated at a cell density of 6,300/cm²,resulted in small, round, non-adherent cells. Non-sorted chorion-derivedcells, plated at a cell density of 6,300/cm², resulted in small, round,non-adherent cells. Non-sorted chorion-derived cells plated at a celldensity of 62,500/cm², resulted in small, round, non-adherent cells.

6.3 Example 3 Differentiation of Placental Stem Cells

Adherent placental stem cells were differentiated into several differentcell lineages. Adherent placental stem cells were isolated from theplacenta by physical disruption of tissue from anatomical sites withinthe placenta, including the amniotic membrane, chorion, placentalcotyledons, or any combination thereof, and umbilical cord stem cellswere obtained by physical disruption of umbilical cord tissue.

Placental stem cells and umbilical cord stem cells were established in amedium containing low concentrations of fetal calf serum and limitedgrowth factors. Flow cytometry analysis showed that placental stem cellstypically exhibited a CD200⁺ CD105⁺ CD73⁺ CD34⁻ CD45⁻ phenotype atpercentages of ≧70%. Placental stem cells were found to differentiatedown the adipocyte, chondrocyte and osteocyte lineages.

In an induction medium containing IBMX, insulin, dexamethasone andindomethacin, placental stem cells turned into fat laden adipocytes in 3to 5 weeks. Under osteogenic induction culture conditions, placentalstem cells were found to form bone nodules and have calcium depositionsin their extracellular matrix. Chondrogenic differentiation of PDACs wasperformed in micropellets and was confirmed by formation ofglycosaminoglycan in the tissue aggregates.

6.4 Example 4 Immunomodulation Using Placental Stem Cells

Placental stem cells possess an immunomodulatory effect, includingsuppression of the proliferation of T cells and natural killer cells.The following experiments demonstrate that placental stem cells have theability to modulate the response of T cells to stimulation in twoassays, the mixed lymphocyte reaction assay and the regression assay.

6.4.1 Mixed Lymphocyte Reaction Assays.

The MLR measures the reaction of an effector population against a targetpopulation. The effectors can be lymphocytes or purified subpopulations,such as CD8⁺ T cells or NK cells. The target population is eitherallogeneic irradiated PBMCs, or as in the present studies, mature DCs.The responder population consists of allo-specific cells, estimated at20% of total T cells. The modified placental stem cell MLR usesplacental stem cells in the reaction.

Placental stem cells were plated in 96 well plate wells, and theeffector population was added. Placental stem cells and5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) stainedeffectors were preincubated for 24 hours before targets, mature DCs,were added. After six days, supernatants and non-adherent cells wereharvested. Supernatants were analyzed by Luminex bead analysis, and thecells were analyzed by flow cytometry.

Classically, the MLR produces a proliferative response in both the CD8⁺and the CD4⁺ T cell compartment. This response is a naïve T cellresponse, as two allogeneic donors have never encountered each otherbefore. Both CD4⁺ T cells and CD8⁺ T cells proliferated vigorously inthe standard MLR. When placental stem cells were added to the MLR, theCD4 and CD8 T cell proliferation, as measured by the percentage ofCFSE^(Low) responder cells, was dampened.

The effect of adding placental stem cells to an MLR (PMLR) can be seenin FIGS. 10A and 10B (PMLR trace) and FIG. 11. The results were similarwhether only CD4⁺ or CD8⁺ T cells were used individually, or whetherequal amounts of CD4⁺ T cells and CD8⁺ T cells were used together.Placental stem cells obtained from the amnion-chorion or umbilical cordstroma suppressed the MLR to similar extents, and no difference insuppression was seen between CD4⁺ T cells and CD8⁺ T cells. This wasalso true for the bulk T cell reactions.

A separate MLR was performed using CD4⁺ T cells, CD8⁺ T cells, or bothCD4⁺ and CD8⁺ T cells, and allogeneic dendritic cells (DC). Placentalstem cells were added to the MLR, and the degree of proliferation of theT cells was assessed, using an MLR without placental stem cells as acontrol.

CD4⁺ and CD8⁺ T cells, and CD14⁺ monocytes, were isolated from buffycoats using Miltenyi MACS columns and beads, used according tomanufacturer instructions. Dendritic cells were obtained by a six-dayculture of monocytes in RPMI 1640 supplemented with 1% donor plasma,IL-4, and GM-CSF, and a two-day culture in RPMI 1640 supplemented withIL-1β, TNF-α, and IL-6. Allogeneic T cells and DC in the ratio T:DC of10:1 were incubated to produce a classic 6 day MLR. T cell proliferationwas assessed by staining T cells with CFSE (Carboxy-fluoresceindiacetate, succinimidyl ester) before being added to the assay. CSFE isused to assess the degree of proliferation by measurement of dilution ofthe stain among daughter cell populations.

To this assay, placental stem cells (PSCs) were added at the ratioT:DC:PSC of 10:1:2. The reaction was set up in a 96-well plate in afinal volume of 200 μL RPMI 1640 supplemented with 5% pooled human serum(R5). After six days, non-adherent cells were briefly resuspended andtransferred to a 5 mL tube washed with RPMI, and stained with CD4 andCD8 antibody. Proliferation of the CD4 and CD8 compartment was assessedon a BD FACS Calibur.

Placental stem cells. Placental stem cells were obtained as described inExamples 1 and 2, above. Placental stem cells were obtained from thefollowing placental tissues: amnion (AM), or amnion/chorion (AC).Umbilical cord stem cells were obtained from digestion of umbilical cord(UC). Fibroblasts (FB) and bone marrow-derived mesenchymal stem cells(MSCs) were added as controls.

Results. When placental stem cells are added to the MLR, T cellproliferation is dampened (FIG. 11). Placental stem cells used in theexperiments reflected in FIG. 12 were derived from one placenta,designated 61665. For all placental stem cells tested, when either CD4⁺and CD8⁺ T cells but not both were used, the CD4⁺ compartment wassuppressed to a greater degree than the CD8⁺ compartment (FIG. 12A).Suppression by AM and UC placental stem cells of CD4⁺ activation wasroughly equivalent to suppression mediated by MSCs, with a suppressionof about 60%-75%. When the MLR was run using both CD4⁺ and CD8⁺ T cells,placental stem cells suppressed proliferation in the CD4⁺ compartment toa far greater degree than the CD8⁺ compartment (FIG. 12B). Inparticular, CD4⁺ T cell proliferation suppression by AM placental stemcells approached 90%, exceeding the suppression shown by MSCs. Thedifference in suppression between these two compartments was moststriking for AM and AC placental stem cells.

Placental stem cells from different donors suppress T cell proliferationin the MLR to a different degree (FIG. 13). Placental stem cells from adifferent placenta, designated 65450, suppressed CD4⁺ and CD8⁺ T cellproliferation in the MLR differently than placental stem cells fromplacenta 61665. Strikingly, AC and UC PSCs from placenta 65450suppressed T cell proliferation from 80% to 95%, exceeding thesuppression in this assay by MSCs. AC placental stem cells from placenta65450, however, did not suppress T cell proliferation to an appreciabledegree (compare AM placental stem cell suppression in FIG. 12A).

Placental stem cells also suppressed the activity of Natural Killer (NK)cells in the MLR.

6.4.2 Regression Assay.

Placental stem cells were shown in a regression assay to suppress a Tcell response to a B cell line expressing Epstein-Barr virus (EBV)antigens. The regression assay is a recall assay that measures effectorT cell mechanisms brought about by presentation of EBV antigen peptideson MHC Class I and II of EBV-transformed B cells. The assay is performedby mixing T cells with an artificially created transformed B cell line,the lymphoblastoid cell line (LCL) from the same donor. The LCLexpresses nine Epstein-Barr virus antigens that elicit between them arange of adaptive T and B cell responses, although in the classicregression assay, only T cell effector mechanisms are measured. Theregression assay offers a convenient way of measuring cytotoxicity totargets infected with a naturally occurring pathogen, in that the LCLexpresses the activated B cell marker CD23. Therefore, the cell count ofCD23-expressing cells is a measure of the number of LCL surviving in theassay.

The classic seventeen day regression assay gave results similar to thoseseen in the first cluster of bars in FIG. 14. No CD23⁺ cells weredetected, as they had all been killed by CD4⁺ and CD8⁺ T cells. With theaddition of placental stem cells, seen in the next two clusters of bars,survival of CD23⁺ cells was enhanced. Without wishing to be bound bytheory, two explanations can be given for the observed effect. Eitherthe T cells had died, and left behind the LCL to expand freely, orplacental stem cells mainly increased the longevity of the LCL, havinghad less of an effect on the T cells.

In a separate regression assay, T cells and dendritic cells wereobtained from laboratory donors. Epstein-Barr virus-transformed B cellslines, LCLs, were obtained by incubating peripheral blood mononuclearcells (PBMCs) with supernatant from a lytic EBV line, B95.8, andcyclosporin A for two weeks. The LCL expressed 9 EBV antigens. Theoutgrowing LCL line is maintained in RPMI 1640 supplemented with 10%fetal calf serum. The regression assay was performed by mixing CD4⁺ orCD8⁺ T cells with autologous LCL at a ratio T:LCL of 10:1. The assay wasperformed in a 96-well plate in 2004 RPMI 1640 supplemented with 5%pooled human serum (R5). To this assay, placental stem cells are addedin a ratio T:LCL:PSC of 10:1:2. The assay was run for 6, 10, or 17 days.

A six-day regression assay was performed using CSFE-labeled T cells.Placental stem cells from placenta 63450 suppressed T cell proliferationin the regression assay by about 65% to about 97%, a result thatcorresponds to the results for these PSCs in the MLR (FIG. 15). Again,UC and AC lines from placenta 63450 significantly suppressed T cellproliferation, while 63450 AM PSCs did not suppress proliferation.

In a separate experiment, it was determined that natural killer cellswere suppressed in the MLR and regression assays, as well. NK cells,when the MLR or regression assay was run including 50 U/ml IL-2, thesuppressive effect was about 45% (range about 40% to about 65%, SEM 5%).

Placental stem cells are not immunogenic. In no instance was more than5% background T cell proliferation observed against placental stem cellsfrom any donor or any placental anatomical site.

Requirement for cell-to-cell contact. The cytotoxic effect in theregression assay, and allo-recognition in the MLR, both depend on TCR (Tcell receptor):MHC interactions between target and effector cells. Therequirements for cell-to-cell contact in placental stem cell-mediatedsuppression was assessed using a transwell assay. In the assay, an MLRwas conducted in which the T cells and placental stem cells wereseparated by a membrane. As seen in FIG. 16, the higher the number ofplacental stem cells used in the MLR, the higher the reduction ofsuppression, indicating that, particularly at higher densities,placental stem cells (UC) require significant contact with the T cellsto suppress T cell proliferation.

A separate assay confirmed that immunosuppression of T cells byplacental stem cells appears to at least partially involve a solublefactor. To determine whether the placental stem cells mediatedimmunosuppression is dependent on cell to cell contact, transwell assayswere performed in which placental stem cells were placed in an insert atthe bottom of which a membrane allowed passage only of soluble factors.At the floor of the well, separated from the placental stem cells, werethe MLR or T cells alone. In order to determine if an observed effectdepended on the relative dose of placental stem cells, the stem cellswere added at different relative densities to T cells and DCs. Whenumbilical cord placental stem cells were separated from the MLR, thesuppressive effect was partly abrogated. When placental stem cells wereused at densities similar to that used in FIG. 11, the MLR suppressionwas abrogated 75% for CD4⁺ T cells, and 85% for CD8⁺ T cells (FIG. 17,FIG. 18). The suppressive effect was still at 66% when just a quarterdose of placental stem cells were used (UC OP 25), and dropped tobackground levels when 12,500 UC placental stem cells were added. Nochange in suppression with separation using an insert was observed (FIG.17). At 25,000 placental stem cells, despite the still vigoroussuppressive effect, the smallest relative drop in suppression onintroduction of the insert was observed (FIG. 18).

6.5 Example 5 Contact Dependence of Placental Stem CellImmunosuppression Differs from that of Bone Marrow-Derived MesenchymalStem Cells

In an experiment to determine the degree of contact dependency inimmunomodulation, umbilical cord stem cells showed a markedly differentrequirement for cell-to-cell contact for immunomodulation than that ofbone marrow derived stem cells. In particular, placental stem cellsdepended more upon cell-to-cell contact to effect immunomodulation,particularly at higher numbers of placental or mesenchymal stem cells.

Bone marrow-derived stem cells (BMSCs) and umbilical cord stem cells(UC) have different requirements for cell-to-cell contact, depending onthe ratio of adherent cells to T cells in a mixed leukocyte reactionassay (MLR). In a transwell experiment, in which placental stem cellswere separated from T cells and dendritic cells (DCs) in the MLR, thesuppression varied between the two types of adherent cell. FIG. 15displays results from the open well and transwell side by side. Whenapproximately 100,000 or 75,000 UC or BMSCs were used in the open wellformat, a similar suppression was observed. However, in the transwellformat, UCs suppress the MLR to a lesser degree than do BMSCs,indicating a larger contact dependency at these higher placental stemcell/T cell ratios. When lower placental cell to T cell ratios wereused, placental stem cells were more suppressive cell for cell.

From the suppression data, the degree of contact dependency wascalculated. FIG. 19 shows the contact dependency of the UC and BMSCMLRs. Bone marrow-derived cells are less contact dependent at higherBM/T cell ratios than are UCs. In other words, UC placental stem cellsand BMSCs behave differently with respect to an important mechanisticparameter, the need for cell-to-cell contact.

Regulatory T cells (Tregs) are necessary for BMSC-mediated T cellsuppression. See Aggarwal & Pittenger, “Human Mesenchymal Stem CellsModulate Allogeneic Immune Cell Responses,” Blood 105(4):1815-1822(2004). CD4⁺CD25⁺ Tregs were depleted from healthy donor peripheralblood mononuclear cells (PBMCs), and a regression assay was performedusing autologous EBV (Epstein-Barr virus)-transformed cells. UCs wereadded to some conditions. As can be seen in FIG. 20, there is nodifference in placental stem cell-mediated suppression of the T cellresponse in the regression assay whether or not Tregs are present. Thus,while T regulatory T cells are reportedly necessary for BMSC-mediated Tcell suppression, T regulatory cells do not appear to play a role inplacental stem cell-mediated immune suppression.

An MLR was performed in which the T cells were taken from an MLRsuppressed by placental stem cells, and the dendritic cells were addedfresh. The T cells were stained with CFSE, which is distributed equallyinto daughter cells during proliferation. CFSE^(Hi) cells are T cellsthat have not proliferated (e.g., the left-most peaks in the panels inFIG. 21). This population was obtained by sorting stained T cells on aFACS Aria. These cells were used in a second MLR with fresh dendriticcells. As can be seen in FIG. 22, no lasting suppression was observed,as the formerly suppressed cells proliferated well against the DCs. Itis unlikely that the CFSE^(Lo) cells (that is, daughter cells) wouldhave been responsible for the suppression, as these cells themselvesproliferated subsequently. The CFSE^(Hi) population is made up ofnon-allo-specific cells that would not have proliferated against this DCdonor, as well as T cells suppressed by placental stem cells. Once theplacental stem cells were removed, the suppressed cells proliferated.

An MLR is suppressed by BMSCs when approximately 10% of the supernatantis replaced by the supernatant from a BMSC MLR. In sharp contrast, nochange in T cell proliferation was observed when supernatant wasreplaced by supernatant from an MLR comprising placental stem cells,even when 75% of the medium was replaced (FIG. 23).

It is possible that DCs or resting T cells are affected by incubationwith placental stem cells for different amounts of time before startingthe MLR. This was tested by incubating placental stem cells or BMSCswith T cells (FIG. 24A) or DCs (FIG. 24B) for varying lengths of timebefore starting the assay. Preincubating T cells and placental stemcells does not alter the suppressive phenotype appreciably (FIG. 24A).However, BMSC T cell suppression changes depending of the length ofDC/PDAC preincubation. As shown in FIG. 24B, suppression by BMSCs isstrongest when DCs are added one day after the T cells. A much lowersuppression appears, however, when DCs are added at the same time as Tcells. Incubating DCs longer with BMSCs can reverse this loss ofsuppression. At two days preincubation, the suppression approaches thescenario where DCs are added a day after T cells (+1 day). No similartendency is observed with placental stem cell-mediated suppression.

6.6 Example 6 Cytokine Profile of Placental Stem Cells and UmbilicalCord Stem Cells in the MLR and Regression Assay

Umbilical cord stem cells (UC) and placental stem cells from amnionchorion plate (AC) were determined to secrete certain cytokines into theMLR medium.

In some assays, a cytokine array was used to measure the levels ofcytokines and chemokines in the supernatants. Several factors were foundto be secreted into supernatants, the most relevant to the MLR andregression assays being macrophage inflammatory protein (MIP)-1α andMIP-1β. Both of these chemoattractants attract T cells, and are secretedby CD8⁺ T cells in response to human immunodeficiency virus (HIV)infection. When assayed in the MLR, these chemoattractants' secretioncorrelated inversely with placental stem cell and MSC suppression of theMLR (FIG. 25). Neither placental stem cells nor MSCs secreted MIP-1α andMIP-1β.

In another study, a correlation was found in secretion of MCP-1 andIL-6, both of which are important immuno-regulators (FIG. 26 and FIG.27; compare with FIG. 11). While placental stem cells alone secreted noIL-6 or MCP-1, the UC and AC lines, both of which suppress the MLR and Tcell proliferation in the regression assay (FIG. 11), secrete MCP-1 andIL-6 (FIG. 26 and FIG. 27). Although IL-6 is mostly associated withpro-inflammatory actions (see, e.g., Kishimoto et al., Annu. Rev.Immunol. 23:1-21 (2005)), it also has other functions, such as aprotective role during liver damage in mice (see, e.g., Klein et al. J.Clin. Invest 115:860-869 (2005)).

In a separate study, AC used in an MLR or regression assay were analyzedfor cytokine secretion. Cytokines were measured on a Luminex system insupernatants from 6-day stem cell cultures, stem cell MLRs or stem cellregression assays. MLRs included the stem cells, dendritic cells (DC),and T cells in a ratio of 2/1/10. Epstein-Barr virus (EBV) regressionassays included stem cells, EBV tumor cells (Ts), and T cells at TS:stemcell:T ratio of 2:1:10.

Levels of IL-6 (11 ng/ml) and IL-8 (16 ng/ml) were found to stayconstant in stem cell solo cultures, MLRs, and regression assays. Theconcentration of MCP-1 was determined to be about 2 ng/ml in stem cellsolo cultures and non-suppressive control adherent cell MLRs andregression assays, but increased to about 10 ng/ml in suppressed stemcell MLRs and stem cell regression assays. These values fall withinserum levels recorded for MCP-1.

Interleukin-2 (IL-2) is both a T cell survival factor and an obligatefactor for CD4⁺CD25⁺ T regulatory cells. This T cell subset is notrequired for T cell suppression by the AC stem cells, but IL-2 levelsconsistently decrease during MLR suppression by AC stem cells. MLRsupernatants in the absence of AC stem cells contained about 35 pg/mlIL-2, whereas the MLRs that included AC stem cells contained up to 440μg/ml IL-2.

The IL-2 concentrations correlated with suppression. For example, a CD4⁺T cell MLR showing 85% suppression contained 330 μg/ml IL-2, and a CD8⁺T cell MLR showing 85% suppression, using AC stem cells contained 66μg/ml IL-2. These results indicate that IL-2 and MCP-1, traditionallyknown as stimulators of the immune response, may play a role in immunesuppression.

EQUIVALENTS

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described will become apparent to thoseskilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various publications, patents and patent applications are cited herein,the disclosures of which are incorporated by reference in theirentireties.

1. A method of treating an individual having multiple sclerosiscomprising administering a therapeutically effective amount of placentalstem cells to the individual in an amount sufficient to detectablyimprove one or more symptoms of multiple sclerosis in the individual,wherein said placental stem cells detectably suppress T cellproliferation stimulated by Epstein-Barr virus antigen-presenting Bcells; and wherein at least 80% of said placental stem cells expressCD200.
 2. The method of claim 1, comprising administering 1 million to10 billion placental stem cells to said individual.
 3. The method ofclaim 2, comprising administering 1×10⁶ placental stem cells to saidindividual.
 4. The method of claim 2, comprising administering 1×10⁷placental stem cells.
 5. The method of claim 2, comprising administering1×10⁸ placental stem cells.
 6. The method of claim 2, wherein said atleast 80% of said 1 million to 10 billion said placental stem cells areOCT-4⁺.
 7. The method of claim 2, wherein said at least 80% of said 1million to 10 billion said placental stem cells are one or more ofCD34⁻, CD38⁻, or CD45⁻.
 8. The method of claim 6, wherein said at least80% of said 1 million to 10 billion said placental stem cells are one ormore of CD34⁻, CD38⁻, or CD45⁻.
 9. The method of claim 2, wherein saidat least 80% of said 1 million to 10 billion said placental stem cellsare CD10⁺ or CD105⁺.
 10. The method of claim 6, wherein said at least80% of said 1 million to 10 billion said placental stem cells are CD10⁺or CD105⁺.
 11. The method of claim 2, wherein said at least 80% of said1 million to 10 billion said placental stem cells are CD10⁺, CD34⁻,CD105⁺ and CD200⁺.
 12. The method of claim 1, wherein said one or moresymptoms comprises or more of a sensory disturbance in the limbs, anoptic nerve dysfunction, a pyramidal tract dysfunction, a bladderdysfunction, a bowel dysfunction, a sexual dysfunction, ataxia, ordiplopia.
 13. The method of claim 1, wherein said individualdemonstrates and improvement on the Expanded Disability Status Scale ofat least one point.
 14. The method of claim 1, wherein said individualdemonstrates and improvement on the Expanded Disability Status Scale ofat least two points.
 15. The method of claim 1, wherein said individualhas relapsing/remitting multiple sclerosis or progressive/relapsingmultiple sclerosis.
 16. The method of claim 1, wherein said individualhas secondary progressive multiple sclerosis or primary progressivemultiple sclerosis.
 17. The method of claim 1, wherein said placentalstem cells are administered during an acute attack of multiple sclerosisin said individual.
 18. The method of claim 1, comprising administeringa second type of stem cell to said individual.
 19. The method of claim18, wherein said second type of stem cell is mesenchymal stem cells frombone marrow.
 20. The method of claim 1, additionally comprisingadministering to said individual a therapeutic agent, wherein saidtherapeutic agent is adrenocorticotropic hormone (ACTH),methylprednisolone, dexamethasone, IFNβ-1a, IFN-1b, gliatriamer acetate,cyclophosphamide, methotrexate, azathioprine, cladribine, cyclosporine,mitoxantrone, or sulfasalazine.
 21. The method of claim 1, wherein saidplacental stem cells have been cryopreserved prior to saidadministration.
 22. The method of claim 1, wherein said placental stemcells have been passaged no more than 10 times prior to saidadministration.